Model for studying the role of genes in tumor resistance to chemotherapy

ABSTRACT

The invention provides the components of in vivo and in vitro systems and methods which use them to study the effects of altered expression of a gene activity, such as the human akt, bcl-2, eIF4E or PTEN activities, on the descendants of stem cells that have been engineered to give rise to hematopoietic tumorigenic or tumor cells, such as lymphomas, with a high frequency. The present invention provides vectors, cells and mammals, and methods which in part depend on such products, useful for understanding tumorigenesis and its treatments, and in particular, for identifying and studying inhibitors and activators associated with tumor cell growth and growth inhibition, cell death through apoptotic pathways, and changes in apoptotic pathway components that affect drug sensitivity and resistance in tumorigenic cells. Methods for identifying molecular targets for drug screening, identifying interacting gene activities, for identifying therapeutic treatments and for identifying candidates for new therapeutic treatments are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to three U.S. Provisional Applications:Ser. No. 60/448,198 filed Feb. 17, 2003; Ser. No. 60/474,742 filed May30, 2003; and Ser. No. 60/______ filed Feb. 4, 2004 (entitled “Model forStudying the Role of Genes in Tumor Resistance to Chemotherapy”), eachof which is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by grants CA87497 andCA13106 from the National Cancer Institute. The Government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention is directed to vectors, cells and mammals, andmethods which in part depend on such products, useful for understandingtumorigenesis and its treatments, and in particular, for identifying andstudying inhibitors and activators associated with tumor cell growth andgrowth inhibition, cell death through apoptotic pathways, and changes inapoptotic pathway components that affect drug sensitivity and resistancein tumorigenic cells.

BACKGROUND OF THE INVENTION

Resistance to cytotoxic agents used in cancer therapy remains a majorobstacle in the treatment of human malignancies, including leukemia andlymphoma. Since most anticancer agents were discovered through empiricalscreens, efforts to overcome resistance are hindered by our limitedunderstanding of why these agents are effective. The role of apoptosisin malignancy provides a new explanation for drug sensitivity andresistance. This view suggests that responsive tumors must readilyundergo apoptosis in response to cytotoxic agents and that resistanttumors may have acquired mutations that suppress apoptosis. Also, thefact that apoptosis is controlled by genes raises the prospect that theproblem of drug sensitivity and resistance will be amenable to molecularbiology.

Apoptosis is controlled by a complex network of proliferation andsurvival genes that is frequently disrupted during tumor evolution. Forexample, the phosphatidylinositol 3-kinase (PI3K) pathway integratesreceptor tyrosine kinase signaling with the apoptotic network (Datta, S.R., et al, “Cellular survival: a play in three Akts,” Genes Dev. 13:2905-2927 (1999) and Vivanco, I. and Sawyers, C. L., “Thephosphatidylinositol 3-Kinase AKT pathway in human cancer,” Nat. Rev.Cancer 2: 489-501 (2002)). One mediator of PI3K signaling is the Akt/PKBkinase, which phosphorylates multiple downstream effectors thatultimately produce global changes in cellular physiology. How Aktpromotes survival is controversial, but may involve directphosphorylation of apoptotic regulators, increased cell cycleprogression, decreased transcription of pro-apoptotic genes viainhibition of forkhead transcription factors, altered metabolism, orchanges in the translation of mRNAs that ultimately control cell death(Vivanco, I. and Sawyers, C. L., “The phosphatidylinositol 3-Kinase AKTpathway in human cancer,” Nat. Rev. Cancer 2: 489-501 (2002)). Mutationsthat activate the PI3K/Akt pathway, including amplifications of PI3Kpathway components and inactivation of the negative regulator PTEN arecommon in human malignancies (Steck, P. A., et al., “Identification of acandidate tumor suppressor gene, MMAC1, at chromosome 10q23.3 that ismutated in multiple advanced cancers,” Nat. Genet. 15: 356-362 (1997);Sakai, A., et al., “PTEN gene alterations in lymphoid neoplasms,” Blood92: 3410-3415 (1998); and Min, Y. H., et al., “Constitutivephosphorylation of Akt/PKB protein in acute myeloid leukemia: itssignificance as a prognostic variable,” Leukemia 17: 995-997 (2003)).Therefore, pharmacological agents that inhibit Akt or its crucialdownstream effectors may be effective against many human cancers.

Evading apoptosis is considered a hallmark of cancer because mutationsin apoptotic regulators invariably accompany tumorigenesis (Hanahan, D.and Weinberg, R. A., “The hallmarks of cancer,” Cell 100: 57-70 (2000)).Many chemotherapeutic agents induce apoptosis, and so disruption ofapoptosis during tumor evolution can promote drug resistance (Johnstone,R. W., et al., “Apoptosis: a link between cancer genetics andchemotherapy,” Cell 108: 153-164 (2002)). Akt is an apoptotic regulatorthat is activated in many cancers and may promote drug resistance invitro (Mayo, L. D., et al., “PTEN protects p53 from Mdm2 and sensitizescancer cells to chemotherapy,” J. Biol. Chem. 277: 5484-5489 (2002)).Nevertheless, how Akt disables apoptosis and its contribution toclinical drug resistance is unclear.

Although Akt has been recognized as both a likely determinant of theoutcome of cancer therapy and a promising therapeutic target (Hsu, J.,et al., Blood, 2001, 98(9): 2853-5; Borlado, L. R., et al., FASEB J,2000, 14(7): 895-903; Brennan, P., et al., Oncogene, 2002, 21(8):1263-1271; Carey, G. B. and D. W. Scott, J. Immunol, 2001, 166(3):1618-1626; Hideshima, T., et al., Oncogene, 2001, 20(42): 5991-6000;Hyun, T., et al., Blood, 2000, 96(10): 3560-3568; Roymans, D. and H.Slegers, Eur J Biociem, 2001, 268(3): 487-498), pharmacologicalinhibitors of Akt are not yet available. However, one of its downstreameffectors, mTOR (mammalian target of rapamycin), can be targeted withrapamycin or its ester CCI-779, which is in clinical trials against avariety of malignancies. It is important to target this pathway forcancer therapies, and to use a valid model to test these therapies andthe nature of their action.

One of the difficulties in identifying determinants of drug cytotoxicityof tumor cells in vivo is the limited availability of appropriatematerial. Human tumor lines grown as xenographs are unphysiological, andthe wide variation between human individuals, not to mention treatmentprotocols, makes clinical studies difficult. Consequently, oncologistsare forced to perform correlative studies with a limited number ofhighly dissimilar samples, often leading to confusing and unhelpfulresults.

Animal models provide a useful alternative to studies in humans and tohuman tumor cell lines grown as xenographs, as large numbers ofgenetically-identical individuals can be treated with identicalregimens. Moreover, the ability to introduce into the mouse germlinemutations which affect oncogenesis increases the power of mouse models.

SUMMARY OF THE INVENTION

The invention provides the components of in vivo and in vitro systemsand methods which use them to study the effects of altered expression ofa gene activity, such as the human akt, bcl-2, eIF4E or PTEN activities,on the descendants of stem cells that have been engineered to give riseto hematopoietic tumorigenic or tumor cells, such as lymphomas, with ahigh frequency. The present invention provides vectors, cells andmammals, and methods which in part depend on such products, useful forunderstanding tumorigenesis and its treatments, and in particular, foridentifying and studying inhibitors and activators associated with tumorcell growth and growth inhibition, cell death through apoptoticpathways, and changes in apoptotic pathway components that affect drugsensitivity and resistance in tumorigenic cells. Methods for identifyingmolecular targets for drug screening, identifying interacting geneactivities, for identifying therapeutic treatments and for identifyingcandidates for new therapeutic treatments are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B together are a diagram of the scheme used to producemice that can be used to study the effect of a gene introduced intolymphomas. In FIG. 1A, hematopoietic stem cells (HSCs) were harvestedfrom the fetal livers of Eμ-myc transgenic mice and retrovirallytransduced with a retrovirus, MSCV, encoding the gene of interest andGFP. Non-transgenic mice were reconstituted with these HSCs followinglethal irradiation. The time between transplantation and manifestationof palpable lymphomas represents the ‘time to onset’. In FIG. 1B, tumorcells were harvested and injected into multiple recipient mice. Upontumor formation, the mice were treated with single agents orcombinations and evaluated for their immediate response, time to relapseand survival.

FIG. 2A is a graph showing time to tumor onset following reconstitutionof the hematopoietic system in non-transgenic C57BL/6 mice with fetalliver cells derived from Eμ-myc transgenic embryos transduced witheither empty parent vector pMSCV-IRES-GFP (n=40, black),pMSCV-Bcl-2-IRES-GFP (n=18, blue), or pMSCV-Akt-IRES-GFP (n=18, green).A kinase-dead mutant Akt (K179A) did not accelerate lymphomagenesis, andAkt did not cause tumor formation in non-transgenic HSCs (data notshown). FIG. 2B shows representative micrographs of Eμ-myc/Akt lymphomasections stained with haematoxylin and eosin (H/E), the rat antibodyagainst mouse CD45R/B220-clone RA3-6B2 (B220), and the antibody againstphosphorylated-Akt (p-Akt).

FIG. 2C shows TUNEL (left) and Ki-67 (right) staining of control, Aktand Bcl-2 lymphoma sections. TUNEL was used to assess apoptosis andKi-67 was used to assess proliferation in cells of the indicatedgenotypes. Extensive staining (in brown) with antibodies to the Ki67antigen in all sections indicating a high rate of proliferation. Incontrast, TUNEL analysis showed much less DNA fragmentation or apoptosisoccurring in Akt or Bcl-2 lymphoma cells relative to control lymphomacells. FIG. 2D shows photomicrographs of H/E stained liver sectionsshowing perivascular infiltration in control tumors and parenchymalinvasion in Akt and Bcl-2 tumors.

FIGS. 2E and F are Kaplan-Meier curves showing the tumor free survivalfollowing treatment with cyclophosphamide (Cytoxan, CTX) (E) or DXR (F).Mice bearing either Arf −/− control lymphomas (black; DXR, n=19; CTX,n=13), Akt lymphomas (green; DXR, n=25; CTX, n=16) or Bcl-2 lymphomas(blue; DXR, n=6; CTX, n=5) were treated and monitored for time torelapse.

FIG. 3 is a Kalpan-Meier curve showing the time of tumor free survivalof mice bearing Akt tumors and Arf −/− control tumors followingtreatment with adriamycin at 10 mg/kg (i.p.) n=20, p=0.0009 (Mantel-Coxtest).

FIG. 4 is a map of the vector pMSCV-Akt-IRES-GFP.

FIG. 5A is a Kaplan-Meier curve showing the proportion of surviving micefor each day after adriamycin treatment. Secondary tumors were producedand treated in mice as described in Example 1. Shown are pooled datafrom the first eight mice in each treatment group, receiving adriamycintreatment. The tumors are from more than five separate primary tumors.FIG. 5B is a Kaplan-Meier curve showing the differences in response toadriamycin treatment according to the genotype of the tumor in thetreated mice. Data from mice were stratified according to the genotypes.

FIG. 5C is a Kaplan-Meier curve showing the response of tumors arisingfrom cells transduced with pMSCV-Bcl-2-IRES-GFP to adriamycin or acombination of adriamycin and rapamycin administered to mice bearing thetumors. FIG. 5D is a Kaplan-Meier curve showing the response of tumorsarising from cells transduced with pMSCV-Akt-IRES-GFP to adriamycin orto a combination of adriamycin and rapamycin administered to micebearing the tumors.

FIG. 6 is a bar graph showing the proportion of mice with secondarylymphomas (produced as in Example 1) that were observed to have acomplete remission following treatment with adriamycin, rapamycin, or acombination of the two.

FIG. 7 is a graph showing the time until relapse (in days) for micetreated for secondary lymphomas produced from seven independentlyarising primary tumors (#135, #136, #92, #93, #102, #103, #104).Treatment regimen was adriamycin alone (10 mg/kg. by i.p. injection) orrapamycin alone (4 mg/kg by i.p. injection), or adriamycin-rapamycin incombination (adr-rapa: adriamycin and rapamycin given on day 1, days 2-5rapamycin alone) or rapamycin-adriamycin in combination (rapa-adr: d1rapamycin, d2 both drugs by separate injection, d3-5 rapamycin).

FIG. 8A shows immunoblots of lysates for phosphorylated and totalribosomal S6 protein (p-S6 and S6), phosphorylated and total eIF4G(p-eIF4G and eIF4G), Akt (p-Akt and Akt), Bcl-2, and a-tubulin (Tub) asa loading control. Animals harbouring control, Akt or Bcl-2 lymphomaswere either left untreated (U) or treated with DXR (A), RAP (R) or DXRand RAP (R+D) and lymphoma cells were harvested 7 hours later. FIG. 8Bshows lymphoma sections derived from untreated or treated animals aslisted above stained with TUNEL (brown) to identify apoptotic cells inthe indicated genotypes.

FIG. 9A is a bar graph representing the percentage of mice bearingcontrol (n=19), Akt (n=51) or Bcl-2 (n=18) expressing lymphomas thatachieved a complete remission (CR) following the indicated treatment.The number of mice in each treatment group ranged from 6 to 25. FIG. 9Bis whole body fluorescence imaging of GFP expression of a ‘matchedgroup’ of mice carrying identical Akt tumors 21 days after the indicatedtreatment. FIG. 9C is a Kaplan-Meier curve showing the tumor freesurvival in Akt lymphomas following treatment with DXR (red, n=25), RAP(blue line, n=12) and RAP+DXR (green, R+D, n=14). FIG. 9D is aKaplan-Meier curve showing (D) Bcl-2 lymphomas following treatment withDXR (n=6; red), RAP (n=6, blue), RAP+DXR (R+D, n=6, green). The tumorfree survival of the control tumors following DXR or CTX treatment isshown for comparison (dotted line).

FIG. 10 is a graph showing the time until relapse (in days) for micetreated for secondary lymphomas produced from eight independentlyarising primary tumors. Mice bearing matched tumors were generated byinjecting 1×10⁶ tumor cells from the same primary tumor into threerecipient animals. Eight ‘matched groups’ of three mice received DXR,RAP, or DXR+RAP. Mice were monitored twice weekly by palpation and bloodsmears. The graph indicates the individual tumor free survival times andthe arithmetic mean; a paired t-test analysis comparing eithercombination to the single agents confirmed statistical significance (forDXR versus RAP+DXR, p=0.0002; RAP versus RAP+DXR, p<0.0001; while DXRversus RAP, p=0.7).

FIG. 11 is a series of Kaplan-Meier curves showing the overall survivalof mice treated with rapamycin alone or in combination with conventionalchemotherapy. Kaplan-Meier analyses of time to death (pre-terminalcondition) following treatment of Eμ-myc/Akt (top panels), E/1-myc/bcl-2(middle panels) and Eμ-myc/eIF4E lymphomas (bottom panel) with RAP(blue), DXR (in A, C and E, red) or CTX (in B and D, red), or thecombinations (green) RAP+DXR (A, C and E) and CTX+RAP (B and D). Thecombination of RAP with DXR or CTX resulted in a prolonged survival inmice bearing Akt expressing tumors (DXR trial, n=51; CTX trial, n=36),but did not extend the survival of mice carrying Bcl-2 or eIF4Eexpressing tumors (Bcl-2 tumors: CTX trial, n=17; DXR trial, n=18; eIF4Etumors, DXR trial, n=17). The survival of Akt tumor bearing mice treatedwith RAP+DXR is indicated as a dotted line in E.

FIG. 12A is a Kaplan-Meier curve showing tumor free survival in Akttumor bearing mice following treatment with CTX (n=16, red), RAP (n=12,blue), or CTX+RAP (C+R, n=8). FIG. 12B is a Kaplan-Meier curve showingtumor free survival in Bcl-2 tumor bearing mice following treatment withCTX (n=6, red), RAP (n=6, blue) and CTX+RAP (C+R, n=4, green). The tumorfree survival of the control tumors following CTX treatment is indicatedin the black dotted line.

FIG. 13A is a Kaplan-Meier curve showing the time to tumor development,following hematopoietic reconstitution with Eμ-myc transgenic HSCsexpressing eIF4E (n=15, p<0.0001 for eIF4E versus pMSCV-control). Thetumor onset curves for HSCs transduced with control pMSCV and Akt areshown for comparison. eIF4E did not induce tumors in a non-transgenicbackground within the observation period (>125 days). FIG. 13B showsrepresentative micrographs of eIF4E lymphoma sections: top panel,haematoxylin and eosin (H/E) staining of the indicated organs; lowerpanel, immunohistochemical stains for phosphorylated Akt, Ki-67 andTUNEL in untreated tumors. FIG. 13C shows immunoblotting of lysatesderived from eIF4E and Akt lymphomas harvested from untreated (U)animals or six hours after rapamycin (R) administration using antibodiesagainst the indicated protein (“p” denotes an antibody specific for aphospho-protein). Untreated Akt tumors (lane 3) show an increase in thephosphorylated, slower migrating forms of 4E-BP1. FIG. 13D shows TUNELstaining (brown) to assess apoptosis in tumor sections harvested 6-8hours following the indicated therapy. FIG. 13E is a Kaplan-Meier curveshowing tumor free survival of mice bearing eIF4E lymphomas followingtreatment with DXR (n=5, red line), RAP (n=6, blue), RAP+DXR (n=6, greenline). Data from mice bearing Akt lymphomas treated with RAP+DXR areshown for comparison (dotted line). FIGS. 13F and G show an in vivocompetition assay. An Akt lymphoma was transduced with MSCV-eIF4E-GFPsuch that the resulting population contained only 2-5% GFP-positivecells and then transplanted into multiple recipient mice. Upon lymphomamanifestation, the animals were left untreated (E) or treated (F) withrapamycin/DXR therapy. Lymphoma cells were isolated 48 hours later andsubjected to flow cytometry to determine the fraction of eIF4E-positivecells (high GFP fluorescence).

FIG. 14 shows the quantification of eIF4E expression in lysates derivedfrom Bcl-2 (n=3), Akt (n=5) and eIF4E (n=5) tumors. (A) Lysates wereprobed with antibodies against eIF4E and b-actin as loading control andI¹²¹ labeled Protein A. Radioactivity was quantitated on Western blotsby phosphorimaging. (B) Graphic representation of eIF4E expressionrelative to Akt and Bcl-2 tumors. The presented data are the combinedaverages of four independent probings of the tumor set with error barsdenoting the standard deviation. Bcl-2 (n=3), Akt (n=5), eIF-4E (n=5).

FIG. 15 shows representative data from flow cytometric immunophenotypingof control (Eμ-myc), Bcl-2 (Eμ-myc/bcl-2), Akt (Eμ-myc/akt) and eIF4E(Eμ-myc/eIF4E) tumors. Tumor cells were gated on the basis of forwardscatter and side scatter and GFP positivity. Both Akt and Bcl-2 tumorsdid not express B-cell antigens other than CD45R (B220) but werepositive for CD4, whereas the control and eIF4E tumors had a matureB-cell marker profile. At least three tumors of each genotype wereanalyzed.

FIG. 16 shows allele-specific PCR to detect the wild-type (p53 WT) andmutant allele (p53 Neo) in tumors derived from Eμ-myc/p53+/− HSCs. Shownare representative results from two tumors per group, these are eithercontrol (C), Akt, Bcl-2 or eIF4E tumors. All control tumors showed lossof heterozygosity (LOH) (8/8), while none of the Akt (0/5), Bcl-2 (0/5)or eIF4E (0/3) tumors underwent LOH.

FIGS. 17A and 17B are two Kaplan-Meier curves showing the survival timefollowing treatment with CTX (A) and DXR (B). Mice bearing eithercontrol lymphomas (black; DXR, n=19; CTX, n=13), Akt lymphomas (green;DXR, n=25; CTX, n=16) or Bcl-2 lymphomas (blue; DXR, n=6; CTX, n=5) weremonitored for time to terminal disease or death.

FIG. 18 is a Kaplan-Meier curve showing the survival time from birth totumor development in mice from a cross of a transgenic Eμ-myc mouse to aPTEN +/− mouse. The control group is Eμ-myc/PTEN +/+ (green line, n=24)and the experimental group Eμ-myc/PTEN +/− (green line, n=12). PTENheterozygosity causes a significant acceleration and increasedpenetrance in lymphoma development in vivo (p<0.05).

FIG. 19A is a Kaplan-Meier curve showing tumor free survival of C57BL/6mice bearing E/1-myc/PTEN +/− tumors following treatment withdoxorubicin (DXR), rapamycin (RAP), and the combination of both(RAP+DXR). Tumor free survival implies the complete absence ofdetectable disease, i.e. no frank leukemia and no palpable tumors, arelapse denotes the recurrence of either. FIG. 19B is a Kaplan-Meiercurve showing an equivalent analysis for treatment with cyclophosphamide(CTX), rapamycin (RAP) and the combination (RAP+CTX).

FIG. 20 shows results from flow cytometry in Eμ-myc/PTEN +/− tumor cellsinfected with eIF4E 48 hours after in vivo therapy. The x-axis indicatesthe GFP fluorescence, denoting tumor cells that are productivelyinfected with MSCV-eIF4E-IRES-GFP. In vivo therapy was a singleinjection of either doxorubicin (DXR) or rapamycin (RAP) or acombination of both (RAP+DXR). An observed increase in GFP-positivecells is noted in all treated tumors as compared to the untreatedcontrol.

FIG. 21 is a Kaplan-Meier curve showing time to tumor development invarious eIF4E mutants. Hematopoietic stem cells were harvested from thefetuses of a pregnant Eμ-myc mouse (ED13-15) and infected in vitro withMSCV-IRES-GFP constructs expressing different mutants of eIF4E,including E103A, S209A, W56A, and W73A.

FIG. 22 is a Kaplan-Meier curve showing the time to tumor development inEμ-myc/short hairpin PTEN (shPTEN) mice following reconstitution oflethally irradiated mice with HSCs from the fetal livers of infectedwith a short hairpin against PTEN (MSCV-shPTEN-IRES-GFP). The controlgroup are equivalent HSCs infected with a vector encoding only GFP(controls; n=25, shPTEN, n=5).

FIG. 23 is an immunoblot showing down-regulation of PTEN expression inthe presence of PTEN short hairpins. Wild type Mouse Embryo Fibroblasts(wt MEFs) containing a control vector (V) or a short hairpin against twomPTEN sequences (PTEN sh1 and PTEN sh2) were collected and analyzed forthe expression of endogenous total and phosphorylated PTEN by Westernblotting.

FIG. 24 shows immunofluorescent staining of Eμ-myc/PTEN +/− tumor cellsincubated with 10 nM rapamycin and incubated with antibodies toS6-kinase. DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) wasadded with the antibody to stain all cells (blue stain on top panels).Untreated cells fluoresced brightly (left), but cells treated withrapamycin treatment (right) showed a marked inhibition of S6-kinase (redstaining).

FIG. 25 is a Kaplan-Meier curve showing tumor free survival ofEμ-myc/p48/eIF3E +/− mice following reconstitution of lethallyirradiated mice with HSCs from the fetal livers of infected with p48fragment of eIF3E (MSCV-eIF3E-IRES-GFP). The control group areequivalent HSCs infected with a vector encoding only GFP (controls;n=25, eIF3E; n=16).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, cell and cancer biology, virology,immunology, microbiology, genetics and protein and nucleic acidchemistry described herein are those well known and commonly used in theart.

The methods and techniques of the present invention are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992, and Supplements to 2003); Harlow andLane Antibodies: A Laboratory Manual Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1990); Coffin et al. Retroviruses, ColdSpring Harbor Laboratory Press; Cold Spring Harbor, N.Y. (1997); Bast etal., Cancer Medicine, 5th ed., Frei, Emil, editors, BC Decker Inc.,Hamilton, Canada (2000); Lodish et al., Molecular Cell Biology, 4th ed.,W. H. Freeman & Co., New York (2000); Griffiths et al., Introduction toGenetic Analysis, 7th ed., W. H. Freeman & Co., New York (1999); Gilbertet al., Developmental Biology, 6th ed., Sinauer Associates, Inc.,Sunderland, Mass. (2000); and Cooper, The Cell—A Molecular Approach, 2nded., Sinauer Associates, Inc., Sunderland, Mass. (2000).

All publications, patents and other references mentioned herein areincorporated by reference.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein, the term “hematopoietic tumorigenic [or tumor] cell”refers to a cell which is or which may become committed to ahematopoietic lineage and which exhibits an altered growth phenotype.The term encompasses cells which are associated with hematopoieticmalignancy (i.e., lymphomas, leukemias) as well as with pre-malignantconditions such as lymphoproliferative and myeloproliferative disorders.

As used herein, the term “hematopoietic stem cell” refers to anymultipotent cell which can produce committed and/or differentiated cellsof the hematopoietic lineage. Hematopoietic stem cells include myeloid(including erythroid, granulocytic and megakaryocytic) and lymphoidprecursor cells and cells not yet committed to either lineage.

As used herein, the term “well defined genotype” refers to a cell of theinvention characterized by comprising a defined combination of geneticlesions. A preferred cell with a “well defined genotype” is one having,e.g., activated myc in combination with one or more activated akt, bcl-2and eIF4E activities (or repressed PTEN or 4E-BP activity). Such lesionsare typically introduced into a cell, individually or in any desiredcombination, by means of DNA (vector)-mediated gene transfer.

As used herein, the term “nucleic acid or “nucleic acid molecule” refersto a polymeric form of nucleotides of at least 10 bases in length. Theterm includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) andRNA molecules (e.g., mRNA or synthetic RNA molecules), as well asanalogs of DNA or RNA containing non-natural nucleotide analogs,non-native internucleotide bonds, or both. The nucleic acid can be inany topological conformation. For instance, the nucleic acid can besingle-stranded, double-stranded, triple-stranded, quadruplexed,partially double-stranded, branched, hairpinned, circular, or in apadlocked conformation.

As used herein, the term “mutation” refers to any change in the nucleicacid or amino acid sequence of a gene product. The term “mutated” whenapplied to nucleic acid sequences means that nucleotides in a nucleicacid sequence may be inserted, deleted, rearranged or otherwise changedcompared to a reference nucleic acid sequence. A single alteration maybe made at a locus (a point mutation) or multiple nucleotides may beinserted, deleted or changed at a single locus. In addition, one or morealterations may be made at any number of loci within a nucleic acidsequence. A nucleic acid sequence may be mutated by any method known inthe art including but not limited to in vivo and in vitro mutagenesistechniques such as “error-prone PCR” (a process for performing PCR underconditions where the copying fidelity of the DNA polymerase is low, suchthat a high rate of point mutations is obtained along the entire lengthof the PCR product. See, e.g., Leung, D. W., et al., Technique, 1, pp.11-15 (1989) and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2,pp. 28-33 (1992)); and “oligonucleotide-directed mutagenesis” (a processwhich enables the generation of site-specific mutations in any clonedDNA segment of interest. See, e.g., Reidhaar-Olson, J. F. & Sauer, R.T., et al., Science, 241, pp. 53-57 (1988)).

As used herein, the phrase “degenerate variant” of a reference nucleicacid sequence encompasses nucleic acid sequences that can be translated,according to the standard genetic code, to provide an amino acidsequence identical to that translated from the reference nucleic acidsequence.

The nucleic acids (also referred to as polynucleotides) of thisinvention may include both sense and antisense strands of RNA, cDNA,genomic DNA, and synthetic forms and mixed polymers of the above. Theymay be modified chemically or biochemically or may contain non-naturalor derivatized nucleotide bases, as will be readily appreciated by thoseof skill in the art. Such modifications include, for example, labels,methylation, substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such asuncharged linkages (e.g., methyl phosphonates, phosphotriesters,phosphoramidates, carbamates, etc.), charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g.,polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.) Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

The term “vector” as used herein is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Other vectors include cosmids, bacterial artificialchromosomes (BAC) and yeast artificial chromosomes (YAC). Anotherpreferred type of vector is a viral vector, wherein additional DNAsegments may be ligated into a viral genome that is usually modified todelete one or more viral genes. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced(e.g., vectors having an origin of replication which functions in thehost cell). Other vectors can be integrated stably into the genome of ahost cell upon introduction into the host cell, and are therebyreplicated along with the host genome. Preferred viral vectors includeretroviral and lentiviral vectors. Moreover, certain preferred vectorsare capable of directing the expression of nucleic acid sequences towhich they are operatively linked. Such vectors are referred to hereinas “recombinant expression vectors” (or simply, “expression vectors”).

“Operatively linked” expression control sequences refers to a linkage inwhich the expression control sequence is contiguous with the gene ofinterest to control the gene of interest, as well as expression controlsequences that act in trans or at a distance to control the gene ofinterest.

The term “expression control sequence” as used herein refers topolynucleotide sequences which are necessary to affect the expression ofsequences (e.g., coding sequences) to which they are operatively linked.Expression control sequences are sequences which control thetranscription, post-transcriptional events and translation of nucleicacid sequences. Expression control sequences include transcriptioninitiation sequences (i.e., promoter and enhancer sequences) whichcontrol the position and rate of transcription initiation. In addition,expression control sequences include appropriate transcriptiontermination sequences, efficient RNA processing signals such as splicingand polyadenylation signals; sequences that stabilize nuclear and/orcytoplasmic mRNA; sequences that enhance translation efficiency (e.g.,ribosome binding sites); sequences that enhance protein stability; andwhen desired, sequences that enhance protein secretion. The nature ofsuch control sequences differs depending upon the host organism. Theterm “control sequences” is intended to include, at a minimum, allcomponents whose presence is essential for expression, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences, stabilization sequences and fusion partnersequences.

The term “overexpression” as used herein with reference to a nucleicacid sequence refers to a higher level of transcription and/ortranslation of a nucleic acid or protein product encoded by a nucleicacid sequence in a cell. Overexpression is most commonly accomplished byoperative linkage of nucleic acid sequences to a strongpromoter/enhancer sequence which stimulates transcription in the targethost cell. Overexpression can be non-regulated (i.e., a constitutive“on” signal) or regulated (i.e., the “on” signal is induced or repressedby another signal or molecule within the cell).

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refers to a cell into which a recombinant vectorhas been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell which resides in a living tissueor organism.

The term “peptide” as used herein refers to a short polypeptide, e.g.,one that is typically less than about 50 amino acids long and moretypically less than about 30 amino acids long. The term as used hereinencompasses analogs and mimetics that mimic structural and thusbiological function.

The term “polypeptide” encompasses both naturally-occurring andnon-naturally-occurring proteins, and fragments, mutants, derivativesand analogs thereof. A polypeptide may be monomeric or polymeric.Further, a polypeptide may comprise a number of different domains eachof which has one or more distinct activities.

As used herein, the term “molecule” means any compound, including, butnot limited to, a small molecule, peptide, protein, sugar, nucleotide,nucleic acid, lipid, etc., and such a compound can be natural orsynthetic.

As used herein, the term “gene X activity” refers to the biologicalactivity encoded by the “gene X” (e.g., akt, bcl-2, eIF4E, PTEN, myc orthe like), and to that activity encoded by a homologous gene or geneproduct from another organism or by an unrelated gene or gene productfrom the same or different organism. Biological activity may be measuredby monitoring any physiological function or attribute of “gene X”. Itmay include enzymatic assays, binding assays, immunoassays, structuraland chemical assays (e.g., which measure modifications such asphosphorylation status, conformational changes) and the like.

As used herein, the term “altered activity” refers to a change in thelevel of a gene and/or gene product with respect to any one of itsmeasurable activities in a cell (e.g., the function which it performsand the way in which it does so, including chemical or structuraldifferences and/or differences in binding or association with otherfactors). An altered activity may be effected by one or more structuralchanges to the nucleic acid or polypeptide sequence, a chemicalmodification, an altered association with itself or another cellularcomponent or an altered subcellular localization. An altered activitymay thus be “activated” or “increased”, “repressed” or “decreased”, ormay remain “normal” (albeit at altered levels), An “activated” or“increased” activity refers, e.g., to overexpression of an encodingnucleic acid, an altered structure (e.g., primary amino acid changes orpost-transcriptional modifications such as phosphorylation) which causeshigher levels of activity, a modification which causes higher levels ofactivity through association with other molecules in the cell (e.g.,attachment of a targeting domain) and the like. Likewise, “repressed” or“decreased” activity refers, e.g., to decreased expression of anencoding nucleic acid (e.g., through antisense or inhibitory RNAiapproaches), an altered structure (e.g., primary amino acid changes orpost-transcriptional modifications such as phosphorylation) which causesreduced levels of activity, a modification which causes reduced levelsof activity through association with other molecules in the cell (e.g.,binding proteins which inhibit activity or sequestration) and the like.

As used herein, the term “hematopoietic stem cell” refers to a precursorcell capable of reconstituting part or all of the hematopoietic systemof a mammal, such stem cells further characterized by their ability toself-renew and to differentiate into cells of the myeloid and/orlymphoid lineages.

An “agent” as used herein can be, for example, a pharmaceutical orchemical with known physiological effects, such as pharmaceuticals thathave been used in chemotherapy for cancer. Agents includechemotherapeutic agents. Chemotherapeutic agents inhibit proliferationof tumor cells, and generally interfere with DNA replication or cellularmetabolism. Chemotherapeutic agents may or may not have beencharacterized for their target of action in cells.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient. Otherchemistry terms herein are used according to conventional usage in theart, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)), incorporatedherein by reference).

“Sensitivity” and “resistance” as used herein refer to the extent towhich tumor growth is inhibited by a treatment. If tumor growth isinhibited by the treatment to a statistically significant degree,compared to a control, the tumor is sensitive to the treatment. If tumorgrowth is not inhibited by the treatment to a statistically significantdegree, compared to a control, the tumor is resistant to the treatment.Tumor sensitivity/resistance can also be relative. For example, a tumorpreviously sensitive to a treatment can become resistant in a relapsedform, where growth of the relapsed form is not inhibited to the sameextent as that seen by the equivalent treatment of the primary tumor.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ofthe present invention and will be apparent to those of skill in the art.

Throughout this specification and embodiments, the word “comprise” orvariations such as “comprises” or “comprising”, will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers. Where the singularterms vector, cell, mammal and mouse are used in the embodiments, theplural meaning is also included, as a single vector, cell, mammal ormouse would not usually be used in isolation.

All publications and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

A description of certain preferred embodiments of the invention follows.

Expressing Akt Signaling Pathway Genes in Hematopoietic Stem Cells

We report an animal model for testing genes involved in apoptoticsignaling pathways—such as the akt gene (encoding the Aktserine-threonine kinase; also known as PKB); bcl-2 gene (encoding b-celllymphoma-2 gene, overexpressed in human follicular lymphoma), eIF4E gene(encoding eukaryotic translation initiation factor 4E); and the PTENgene (encoding a phosphatase with homology to tensin and which is anegative regulator of Akt kinase) for their effects on anti-cancertreatments, the development of drug resistance and treatments toencourage drug sensitivity of tumorigenic cells, especiallyhematopoietic tumorigenic cells.

The function of Akt was investigated by cloning the gene encoding humanAkt1 and expressing an active form of Akt in hematopoietic stem cells.Specifically, Akt 1 was inserted into a murine stem cell virus (MSCV)vector carrying a gene encoding green-fluorescent protein (GFP) whichwas used to infect hematopoietic stem cells that were harvested from thebone marrow or fetal livers of Eμ-myc mice (see, e.g., U.S. Pat. No.6,583,333). These stem cells were used to reconstitute the hematopoieticsystem of lethally irradiated mice (FIG. 1A). Thus, the entirehematopoietic compartment in these mice is derived from the geneticallymodified Eμ-myc stem cells, while the other tissues of the animal arewild type. In these mice the myc transgene is activated during B celldevelopment and causes lymphoma formation. This approach allowed us tostudy the impact of the akt transgene on tumorigenesis (see Example 1).The tumors expressing the transgene are easily identified by their GFPexpression and can be harvested, split, and introduced into multiplenon-transgenic syngeneic mice to form secondary lymphomas (FIG. 1B).This increases the numbers of animals for treatment studies (Schmitt, C.A. and S. W. Lowe, Blood Cells Mol Dis, (2001), 27(1): 206-216, Schmitt,C. A., C. T. Rosenthal, and S. W. Lowe, Nat Med, (2000), 6(9):1029-1035). Using this approach, we found that Akt dramaticallyaccelerates the onset of lymphomas arising in pMSCV-Akt-transducedhematopoietic stem cells taken from Eμ-myc transgenic mice (Example 1;FIG. 2A). In part, this may be due to its anti-apoptotic activity, asAkt expression appears to obviate the need for these tumors to lose thepro-apoptotic p53 gene.

While conferring a selective advantage in vivo, Akt producing B-cellscould not be grown in tissue culture under any of a variety ofconditions tested, underlining the necessity for a suitable in vivomodel. Expression of Akt changes the metabolic requirements of tumorcells. Unlike Bcl-2, which inhibits apoptosis at the mitochondrial leveland confers a general survival advantage, Akt appears to be responsiblefor maintaining mitochondrial membrane potential through increasingglycolysis. See, e.g., Plas, D. R. et al., J. Biol. Chem.276(15):12041-12048, (2001). While Bcl-2 expressing cells grow wellunder any culture conditions, Akt expressing cells are very sensitive toculture conditions. Based on Plas et al. and other reports, we tried avariety of conditions (different concentrations of glucose, 3% oxygen,addition of IL3, IL6, IL7 or nonessential amino acids to the media), butnone of these modifications of the media allowed us to grow Aktexpressing B-lymphoma cells in culture. A pure population ofAkt-expressing cells isolated from a tumor derived by using the stemcell infection and adoptive transfer approach also does not grow inculture at all. In a mixed population of pMSCV-Akt-IRES-GFP infected anduninfected B lymphoma cells, the proportion of GFP positive cellsdropped from between 25% to 35%, depending on the experiment, toundetectable, within 48 hours. This dependence on environmental cuesunderlines the necessity to study Akt in an in vivo model, aspresumably, the only way to keep Akt expressing cells viable in cultureis through accumulation of other genetic defects (loss of p53, etc.)which, while allowing growth in culture, clearly interfere with theiruse as research tools to study the PI3K/Akt pathway and pharmacologicalinterference with it.

To determine how Akt signaling influences the response of tumors tochemotherapy in vivo, the effects of a constitutively activated Aktmutant (Andjelkovic, M., et al., “Role of translocation in theactivation and function of protein kinase B,” J. Biol. Chem. 272:31515-31524 (1997)) to the anti-apoptotic regulator Bcl-2 in the Eμ-mycmodel of B-cell lymphoma (Adams, J. M., et al. “The c-myc oncogenedriven by immunoglobulin enhancers induces lymphoid malignancy intransgenic mice,” Nature 318: 533-538 (1985)), were compared, asdescribed in Example 2. In brief, Eμ-myc hematopoietic stem cellsisolated from fetal livers were transduced with Akt or Bcl-2 expressingretroviruses, transplanted into lethally irradiated mice and thechimeric recipients were monitored for lymphoma onset and pathology(FIG. 1 depicts the method and FIG. 2A charts the results).

Resulting lymphomas were each transplanted into several wild typeanimals, where they produced malignancies that are pathologicallyindistinguishable from the original disease (see Example 1 and Schmitt,C. A. and Lowe, S. W, “Bcl-2 mediates chemoresistance in matched pairsof primary Eμ-myc lymphomas in vivo,” Blood Cells Mol. Dis. 27: 206-216(2001)). As described in Example 2, these animals were then treated witha chemotherapeutic regime and monitored for time to relapse (“tumor freesurvival”) and overall survival by lymph node palpation and bloodanalysis. This approach allows a rapid analysis of genotype-responserelationships in a natural setting using methods that parallel humanclinical trials.

Further, in treatment studies, it has been found that, compared tocontrol tumors (Arf-null tumors used as controls; the response patternsof these tumors are uniquely homogeneous with regard to p53 status), theakt transgene confers a high degree of resistance to cyclophosphamide(FIG. 2E) and adriamycin (FIG. 3). Both are agents central to theconventional therapy of lymphoma Specifically, the Akt producing tumorsrelapse significantly earlier than do the control tumors. Thistranslates into a dramatic reduction in survival, especially for thecyclophosphamide treatment, which is curative for many of the controltumors but none of the Akt tumors.

Vectors

Akt-Vectors

In one aspect, the invention comprises vectors carrying a gene,constructed for high level expression of the gene, starting from vectorsbased on murine stem cell virus (MSCV). The vectors can comprise an aktgene as described in the prior art, or other akt genes to be isolated asnatural variants or produced by manipulation in the laboratory. The Aktprotein produced in cells into which the vector is introduced can havevarying levels of phosphorylation activity, according to the Akt genechosen to be constructed into the vector. A particular vector describedherein is pMSCV-Akt-IRES-GFP, which encodes human Akt and which has theadvantage of carrying a linked marker gene encoding green fluorescentprotein.

One important feature of a vector that can be used to introduce a geneinto recipient cells is a high frequency of integration into therecipient cell genome. Another desirable feature of such a vector is ahigh level of constitutive gene expression for the inserted gene ofinterest. Vectors that are derived from the murine stem cell virus(MSCV) have been developed for studying gene function in hematopoietic,embryonic stem cells and embryonal carcinoma cells. Following insertionof a gene into the vector, the vector is transfected into a packagingcell line to allow for replication of the nucleic acid and packaging ofviruses which are unable to replicate in cells not engineered to produceretroviral proteins. Cell lines for producing amphotropic virus, as wellas cell lines for producing ecotropic virus, are available. For vectorscarrying a human oncogene to be expressed in cells, a cell line toproduce ecotropic virus is desirable for safety concerns. Constructionand propagation of useful MSCV vectors is described, for example, inHawley, R. G. et al., Proc. Natl. Acad. Sci. USA 93:10297-10302 (1996)and in Chen, L. et al., Blood 1:83-92 (1998), and in the catalog andtechnical information from Clontech/BD Biosciences, which suppliespMSCVneo and related pMSCV vectors, along with packaging cell lines.

pMSCV-based vectors carrying a human Akt oncogene are referred to hereas pMSCV-Akt vectors. A pMSCV-Akt vector comprises, at a minimum, avector comprising 5′ and 3′ LTRs of the murine stem cell virus (MSCV),an akt gene with expression driven by the LTR from MSCV, and ψ⁺ forexpressing the extended viral packaging signal (see Example 1 and FIG.4). The akt gene can be a human gene. The akt gene can be wild-type(normal), a mutant or variant found in nature, or an artificiallymutated or modified gene. The gene can encode a gene product differingfrom that of a wildtype gene product, such as an inactive gene productor a gene product conferring a dominant negative phenotype. ThepMSCV-Akt-IRES-GFP vector, cells transduced with it, and animals bearingsuch cells are discussed below as illustrations of a more generalpMSCV-gene vector, in which any gene of interest can be inserted, forexample, an oncogene or a gene suspected to play a role in apoptosis, orin tumor maintenance (see Example 1).

A pMSCV-based vector, pMSCV-Akt-IRES-GFP, was constructed to providehigh level expression of the human akt gene in hematopoietic stem cellsand their progeny that undergo differentiation. Although the combinationof IRES and GFP in the vector allows for the convenient separation,quantitation and in vivo detection of cells transduced with theMSCV-Akt-IRES-GFP vector, either IRES or GFP or both would not beessential elements in a pMSCV-Akt vector. Such a pMSCV-Akt vectorlacking IRES, GFP or both, could be used, for example, in experimentsthat do not require physically locating the transfected cells. Forexample, one could use a pMSCV-Akt vector in a study in which one wouldtest the effectiveness of an agent to inhibit the development oflymphomas, by introducing into two populations of mice a pMSCV-Aktvector, treating one population with a putative anti-lymphoma agent, butnot treating, or sham-treating, the other population, and following thetime until lymphomas were detected.

In one embodiment, the invention provides vectors comprising aktsequences, preferably (but not necessarily) linked to one or moreexpression control sequences which will regulate its expression in ahost cell. In one preferred embodiment of the akt expression vector, aktactivity is activated by cellular localization of the catalytic domainto the plasma membrane of the cell in which it is produced. One knownway for accomplishing plasma membrane localization is through physicalassociation of the Akt polypeptide with a myristoylation/palmitylationmotif which targets the Akt polypeptide to the plasma membrane.Preferably, the myristoylation/palmitylation motif is engineered at theN-terminus of the Akt polypeptide by linkage of appropriate nucleic acidsequences and expression of the hybrid sequences in the selected hostcell. It will be understood that akt activation by cellular localizationcan be accomplished by any means. In another preferred embodiment,plasma membrane localization of akt kinase activity remains subject tophysiological regulation. Further, localization of Akt to the plasmamembrane may be inducible, e.g., through an inducible association with amotif, such as a myristoylation/palmitylation motif, which mediatesplasma membrane localization.

In certain preferred embodiments, the invention provides a vectorcapable of being introduced into a stem cell comprising a nucleic acidsequence which expresses akt activity upon expression in a hematopoieticcell. Preferably, the vector causes the cell in which it is expressed toproduce an altered akt activity, i.e., which is different in phenotype(e.g., having altered activity) and/or having increased or decreasedlevels of “normal” akt activity relative to a control cell lacking theexpressed nucleic acid sequence.

Preferably, an akt vector of the invention comprises nucleic acidsequences derived from mammalian sequences, and more preferably, fromhuman sequences. The akt nucleic acid sequences may be wild-type,variant, derivative (e.g., fused to other domains or chemically altered)or mutated sequences. For example, in certain preferred embodiments, theencoded Akt polypeptide has altered akt biological activity. In certainembodiments, for example, it is preferred that the akt have reduced oreliminated kinase activity. Overexpression of such an akt mutant willhelp modulate the akt signaling pathway and is expected to facilitatethe ability of a tumor cell to regain sensitivity to a drug or otheragent to which it has become chemoresistant through an akt-, bcl-2-,eIF4E-mediated signaling pathway.

Also provided by the invention are vectors which are capable ofintroducing a nucleic acid sequence into stem cells or tumorigenic cellsand which encode sequence specific inhibitors of akt activity.Preferably, the vectors encode a shRNA (short hairpin RNA) whichmediates inhibition of akt signaling in a stem cell, such as ahematopoietic stem cell, or in a tumor or tumorigenic cell, especiallyof a hematopoietic lineage. Such vectors will be useful for making cellsand mammals in which akt signaling is modulated according to the methodsof the invention.

bcl-2-, eIF4E- and PTEN Vectors

In other embodiments, the invention provides a vector capable of beingintroduced into a stem cell comprising a nucleic acid sequence whichexpresses akt, bcl-2, eIF4E or PTEN activity upon expression in ahematopoietic cell. Preferably, the vector causes the cell in which itis expressed to produce an altered akt, bcl-2, eIF4E or PTEN activity,i.e., which is different in phenotype (e.g., having altered activity)and/or having increased or decreased levels of otherwise “normal”activity relative to a control cell lacking the expressed nucleic acidsequence.

The invention further provides vectors comprising bcl-2 nucleic acidsequences which can be introduced into stem cells or tumorigenic cellsand from which bcl-2 sequences can be expressed. Preferably, a bcl-2vector of the invention comprises nucleic acid sequences derived frommammalian sequences, and more preferably, from human or mouse sequences.The bcl-2 nucleic acid sequences may be wild-type, variant, derivative(e.g., fused to other domains or chemically altered) or mutatedsequences. For example, in certain preferred embodiments, the encodedbcl-2 polypeptide has altered biological activity. In certainembodiments, for example, it is preferred that the bcl-2 have reduced oreliminated activity. Overexpression of such a bcl-2 mutant will helpmodulate the akt-/bcl-2-/eIF4E signaling pathway and is expected tofacilitate the ability of a tumor cell to regain sensitivity to a drugor other agent to which it has become chemoresistant through an akt-,bcl-2-, eIF4E-mediated signaling pathway. A particular preferred vectordescribed herein is pMSCV-bcl-2-IRES-GFP, which encodes a human bcl-2and which has the advantage of carrying a linked marker gene encodinggreen fluorescent protein, as described above for akt vectors.

Also provided by the invention are vectors which are capable ofintroducing a nucleic acid sequence into stem cells or tumorigenic cellsand which encode modulators (activators or inhibitors) of bcl-2activity. Preferred modulators include sequence specific inhibitors ofbcl-2. Preferably, the vectors encode a shRNA (short hairpin RNA) whichmediates inhibition of bcl-2 signaling in a stem cell, such as ahematopoietic stem cell, or in a tumor or tumorigenic cell, especiallyof a hematopoietic lineage. Such vectors will be useful for making cellsand mammals in which the akt-/bcl-2-/eIF4E signaling pathway ismodulated according to the methods of the invention.

The invention further provides vectors comprising eIF4E nucleic acidsequences. Vectors can comprise an eIF4E gene as described in the priorart, or other eIF4E genes to be isolated as natural variants or producedby manipulation in the laboratory. The eIF4E protein produced in cellsinto which the vector is introduced can have varying levels of bindingactivity to other components of the multimolecular complexes involved intranslation initiation, resulting in varying rates or levels ofefficiency of translation of mRNAs, according to the eIF4E gene chosento be constructed into the vector. A particular preferred vectordescribed herein is pMSCV-eIF4E-IRES-GFP, which encodes a murine eIF4Eand which has the advantage of carrying a linked marker gene encodinggreen fluorescent protein, as described above for akt vectors.

Another preferred eIF4E vector of the invention comprises a mutation ata phosphorylation site (S209A) which is the target of a kinase (MNK-1 or-2) in the MAP kinase pathway (through MEK). Phosphorylation of S209 wasshown previously not to be inhibited by rapamycin. In contrast, as shownherein, a stem cell transduced with an eIF4E vector comprising the S209Amutation, when introduced into mouse hematopoietic stem cells engineeredto become tumor cells, produces a mouse which exhibits a significantlylonger time to onset of tumor formation than does a mouse expressing acorresponding non-mutated eIF4E vector.

Also provided by the invention are vectors which are capable ofintroducing a nucleic acid sequence into stem cells or tumorigenic cellsand which encode modulators (activators or inhibitors) of eIF4Eactivity. Preferred modulators include sequence specific inhibitors ofeIF4E activity. Preferably, the vectors encode a shRNA (short hairpinRNA) which mediates inhibition of eIF4E signaling in a stem cell, suchas a hematopoietic stem cell, or in a tumor or tumorigenic cell,especially of a hematopoietic lineage. Other preferred vectors encode4E-BP activity (4E-BP1, 2 and/or 3) which bind to eIF4E and therebyinhibit its activity. Such eIF4E vectors will be useful for making cellsand mammals in which the akt-/bcl-2-/eIF4E signaling pathway ismodulated according to the methods of the invention.

The invention further provides vectors comprising PTEN nucleic acidsequences which can be introduced into stem cells or tumorigenic cellsand from which PTEN sequences can be expressed. Inhibition of PTENintroduced into mouse hematopoietic stem cells engineered to becometumor cells produced a mouse which exhibited a significantly reducedtime to onset of tumor formation than did a control mouse (see Example12). Thus, overexpression of PTEN is expected to result in decreased aktsignaling and increased sensitivity to rapamycin in combination withchemotherapeutic agents.

Also provided by the invention are vectors which are capable ofintroducing a nucleic acid sequence into stem cells or tumorigenic cellsand which encode modulators (activators or inhibitors) of PTEN activity.Preferred modulators include sequence specific inhibitors of PTENactivity. Preferably, the vectors encode a shRNA (short hairpin RNA)which mediates inhibition of PTEN and thus akt signaling in a stem cell,such as a hematopoietic stem cell, or in a tumor or tumorigenic cell,especially of a hematopoietic lineage. Two exemplary vectors whichencode shRNAs that mediate PTEN inhibition are described herein (seealso SEQ ID NOS:1 and 2). Such PTEN vectors will be useful for makingcells and mammals in which akt-/bcl-2-/eIF4E signaling is modulatedaccording to the methods of the invention.

In general, a vector of the invention must be capable of beingintroduced into a stem cell, preferably a mammalian stem cell, and morepreferably, a hematopoietic stem cell. The vector may be introduced byany of numerous methods known in the art for introducing nucleic acidsinto eukaryotic cells. See, e.g., Ausubel et al., Current Protocols(supra). In a preferred embodiment, the vector of the invention is aviral vector capable of transducing a mammalian stem cell, andpreferably, a hematopoietic stem cell. A variety of viral vectors areenvisioned and the invention is not limited to the viral vector type aslong as the vector can successfully infect or transduce a stem cell withthe desired activity.

Preferred viral vectors are retroviral based vectors. One preferredretroviral vector is derived from MMLV. Another preferred retroviralvector (used in the Examples of the invention set forth herein) is themurine stem cell virus, “MSCV” (see, e.g., McCurrach and Lowe, MethodsCell Biol. 66:197-227 (2001) for exemplary retroviral methods; see alsoCoffin et al., Retroviruses, supra (1997)). Another preferred viralvector is a lentiviral based vector that can transduce or infect a stemcell, and preferably, a hematopoietic stem cell. Many retroviral andlentiviral vector systems and packaging cell lines are known in the artand available.

Other preferred vectors of the invention comprise a nucleic acidsequence which expresses akt, bcl-2, eIF4E or PTEN activity uponexpression in a hematopoietic cell wherein part or all of the nucleicacid sequence encoding such activity can be deleted or excised fromsurrounding sequences even after it has integrated into the host genome.Any of a number of sequences which mediate excision from DNA uponexpression of a cognate recombinase and corresponding recombinaseactivities may be used to induce such excisions or deletions, e.g.,cre-lox recombinase/lox-P sites; Flp recombinase/Frt sites; andpfiC31/att sites. A nucleic acid sequence encoding akt, bcl-2, eIF4E orPTEN activity is flanked by sequence motifs that mediate deletion ofinternal sequences upon expression of the cognate recombinase activity.Preferably, the recombinase is operatively linked to an induciblepromoter. Thus, for example, the invention provides, e.g., acre-estrogen receptor fusion protein induced by tamoxifen to delete thetransduced gene so that induction causes recombinase-mediated excisionof akt, bcl-2, eIF4E or PTEN-encoding sequences from the genome.Alternatively, a vector containing the loxP sites flanking the gene maybe introduced into stem cells harboring a transgene that conditionallyexpresses cre recombinase. In either case, upon tumor formation, theintroduced gene may be deleted. As discussed supra, reduction orelimination of akt, bcl-2 or eIF4E biological activity will increase thesensitivity of a tumor cell to certain therapeutic treatments to which atumor cell has acquired resistance through activation of an akt-,bcl-2-, and/or eIF4E-mediated signaling pathway.

Various embodiments of the invention involve expressing akt signalingpathway components having altered (e.g., increased or decreased)activity. As shown using the mouse models for tumorigenesis describedherein, increasing activity of akt-, bcl-2 or eIF4E or decreasingactivity of the akt inhibitor PTEN leads to activated akt signaling andincreased chemoresistance, whereas decreasing activity of akt-, bcl-2 oreIF4E or increasing activity of the akt inhibitor PTEN leads todecreased akt signaling and increased chemosensitivity. Thus, forpurposes of producing mammalian models for studying genes (e.g., aspotential therapeutic targets), genetic alterations and for screeningpotential drug candidates which can alter chemosensitivity orresistance, it will be desirable to modulate one or more of each ofthese activities in either direction. Modulation of other components ofthe pathway that interact with Akt-, Bcl-2, eIF4E (e.g., 4E-BP) or PTENis also envisioned. Accordingly, also encompassed by the presentinvention are nucleic acid molecules which can effectively modulate(e.g., inhibit) expression of a target gene activity (i.e., akt-, bcl-2,eIF4E or PTEN) in a cell or a mammal of the invention. Preferably,shRNAs (short hairpin RNAs) are used as sequence specific expressioninhibitors using “RNAi” techniques which are well known in the art. See,e.g., Scherer and Rossi, Nature Biotechnology 21:1457-65 (2003) for areview on sequence-specific mRNA knockdown of using antisenseoligonucleotides, ribozymes, DNAzymes, RNAi and siRNAs.

Certain preferred vectors of the invention comprise nucleic acidsequences encoding the activity of interest (akt, bcl-2, eIF4E or PTEN)and further comprise one or more additional gene(s) capable ofexpression. The additional gene(s) may encode one or more of a varietyof functions. For example, it may encode a selectable marker gene whosepresence and/or whose expression co-localizes with the presence and/orexpression of the activity of interest (akt, bcl-2, eIF4E or PTEN). Theadditional gene may encode a detectable marker gene such as afluorescent marker (e.g., GFP, luciferase, color-shifted variants andthe like). In some preferred embodiments, the additional gene may encodean oncogenic activity which contributes to the ability of a cellharboring the vector to become transformed, e.g., into cells havingmalignant or pre-malignant phenotypes, especially of the hematopoieticsystem. As described herein, a mammal that receives cells transducedwith such vectors will develop tumors, especially hematopoietic tumors.In certain preferred embodiments, the oncogene is an activated myc gene,such as one whose expression is operatively linked to a strong viralpromoter and/or enhancer. One such preferred vector comprises the Eμ-mycgene, which is derived from a cellular myc gene activated by insertionof an Ig enhancer. Of course, any other means for activating expressionof the myc gene may be used in the vectors (and cells and mammals) ofthe invention.

In other preferred embodiments, the additional gene may encode asequence specific expression inhibitor, such as one described above.Preferred inhibitors encode one or more shRNAs (short hairpin RNAs) orother inhibitory nucleic acid which, upon expression in a stem cell orprimary hematopoietic tumor cell (e.g., a lymphoma), modulate theactivity of the akt-/bcl-2-/eIF4E signaling pathway thereby affectingtumorigenesis, apoptotic cell death and chemosensitivity and resistance.

In other embodiments, the additional gene may be transcriptionallylinked to nucleic acid sequences encoding the activity of interest (akt,bcl-2, eIF4E or PTEN). If so, it is preferred that they be translatedfrom a downstream internal ribosome entry site (IRES) rather than beingexpressed as part of a hybrid fusion protein. Thus, certain preferredvectors of the invention comprise a nucleic acid sequence encoding anactivity of interest (e.g., akt, bcl-2, eIF4E or PTEN) linked to asequence encoding IRES-gene 2 sequence. Preferred retroviral vectors ofthe invention thus include pMSCV-akt-IRES-gene 2, pMSCV-bcl-2-IRES-gene2, pMSCV-eIF4E-IRES-gene 2, and pMSCV-akt-PTEN-gene 2. It is alsopossible to switch the order of gene 2 such that it is transcribedupstream from (5′ to) an IRES-nucleic acid sequence encoding theactivity of interest (akt, bcl-2, eIF4E or PTEN). Moreover, there is noreason to limit such constructs to bicistronic messages. Tri- ormulti-cistronic mRNAs may be constructed using several IRES sequences intandem and are encompassed in the present invention.

Cells

Vectors of the invention can be introduced into cells, for example, byviral transduction, and a high frequency of integration of the vectorcan be achieved. The cells can be cells of a mammal, preferably a rodentsuch as a rat, more preferably a mouse, and even more preferably, ahuman cell. The cells for study can be chosen for a genotype thatfacilitates study of pathways involved in apoptosis and resistance tochemotherapeutic agents. For example, the cells can be Eμ-myc mousecells, and, in a particular embodiment, are Eμ-myc mouse hematopoieticstem cells. The cells into which the vectors have been introduced areanother aspect of the invention.

The invention provides cells transduced with a pMSCV-Akt vector (e.g.,pMSCV-Akt-IRES-GFP). Transduction procedures are well known in the artand have been described. A transduction procedure is illustrated inseveral of the Examples. Transduced cells integrate thepMSCV-Akt-IRES-GFP vector into the cell genome at a high frequency, andstably express the akt gene. Protein extracted from the transduced cellscan be analyzed for Akt, for example, by western blot, using antibodiesspecific to Akt, as illustrated (e.g., Example 2, Example 7, andExamples 9-11). Transduction and integration of the pMSCV-Akt-IRES-GFPvector allows for tracking of the progeny of the transduced cellsgenerations after the originally transduced cells, by detection of theGFP marker. Other markers which can mark and trace cells and theirprogeny are also encompassed as preferred markers of the invention.

Several of the Examples illustrate the use of cells of a specificgenotype, derived from mice having an activated myc oncogene (e.g.,under the control of the Eμ IgH enhancer). The myc gene can be one asdescribed in Harris, A. W., J. Exp. Med. 167:353-371 (1988) or theallele described by Langdon, W. Y. et al., Cell 47:11-18 (1986), forexample. The myc gene can also be a naturally-occurring gene, eithercellular or viral, a natural variant or an artificially altered variantof myc. Any other oncogene that can accomplish a similar purpose is alsoencompassed as a preferred additional gene of the invention.

Although the use of a pMSCV-Akt vector in mouse hematopoietic stem cellsis described in detail in the Examples, any other type of mammaliancells can be infected with a pMSCV-Akt vector, if an amphotropicretrovirus packaging cell line is used to produce the virus. Theresulting cells can be used in a variety of studies on the effects ofAkt, for example, the effects on the downstream effectors of Akt, theeffect of Akt on apoptosis, cell death, resistance to anti-proliferativetreatments (for example, drugs to be used in cancer chemotherapy) andthe effects of Akt on metabolism and metabolic regulation.

Cells of the invention include any cell into which a vector of theinvention has been introduced. Preferably, the cells of the inventionare mammalian, and more preferably, are rodent (mouse or rat), primateor human. Preferred cells of the invention are stem cells (pluripotentor multipotent), and are, more preferably, hematopoietic stem cells.Also preferred are tumor cells, especially primary tumor cells, intowhich a vector of the invention has been introduced. Cells of theinvention are useful for producing mammals of the invention, especiallyby implantation of stem cells and repopulation of hematopoietic cells ofthe mammal (see below). Certain cells of the invention are prone tobecoming hematopoietic tumor cells. Other cells of the invention aremore resistant to becoming hematopoietic tumor cells. Accordingly, cellsof the invention are also useful as tools in methods for studying cellgrowth and regulation, apoptosis, tumorigenesis, and sensitivity andresistance to chemical agents such as small molecules and otherchemicals which may be useful as drugs in the treatment of diseases,disorders or conditions associated with abnormal regulation of suchcellular processes.

Mammals

In a further aspect of the invention, the cells described above areintroduced into one or more recipient mammals (e.g., mice), therebyproducing trangenic mammals bearing the cells of the invention. Therecipient mammal is engineered to develop hematopoietic tumorigenic ortumor cells such as lymphomas which comprise a gene that modulates theakt-, bcl-2-, eIF4E-, PTEN-, eIF3E- pathway.

Thus, the invention provides a mammal into which a vector or cell of theinvention has been introduced. Preferably, the mammals of the inventionare rodents (e.g., mouse or rat), primates or humans.

We have produced and characterized an animal model for testing theeffects of modulating activity of the akt gene, bcl-2 gene, eIF4E gene,PTEN gene, and eIF3E gene on various anti-cancer treatments, and on thedevelopment of drug resistance in tumor cells. Also included in theinvention are methods for using the mammals of the invention, e.g., micethat develop lymphomas, and the lymphoma cells that arise in the mousemodel, to elucidate the nature of drug resistance, to study the effectsof gene overexpression (or inhibition) on apoptosis-related signalingpathways and on levels of other regulatory factors in the cell.

Thus, a further aspect of the invention is a mammal bearing a populationof cells transduced with a pMSCV-Akt vector such as pMSCV-Akt-IRES-GFP.Mammals bearing such transduced cells can be studied, for example, forthe effect of Akt in apoptosis, in development of resistance to agentsthat promote apoptosis and in treatments to reduce such resistance.

A particular example of mammals bearing a population of cells transducedwith a pMSCV-Akt vector are mice produced by starting with C57BL/6 mice,obtaining fetal liver or bone marrow from them, culturing hematopoieticstem cells from this tissue, and transducing akt into these cells usingpMSCV-Akt-IRES-GFP. The transduced cells are introduced into syngeneicor lethally irradiated C57BL/6 recipient mice, whereupon the transducedcells reseed the bone marrow, thereby producing a population of cellshaving a genome comprising a myc gene operably linked to an Eμ IgHenhancer, and further comprising an akt gene by insertion ofpMSCV-Akt-IRES-GFP into the recipient mouse genome.

The mouse model described herein provides a useful model for studyingapoptosis and cancer therapy, since: (i) tumor burden can be monitoredexternally by lymph node palpation and often by blood smears; (ii)lymphomas are detectable long before the animals die, so animals can betreated while otherwise healthy; (iii) large numbers of tumor cells canbe isolated from mice undergoing therapy or treatment; (iv) therapy isperformed in immunocompetent mice; and (v) lymphoma cells can betransplanted into syngeneic and/or sub-lethally irradiated mice.

Methods

The cells and mammals of the invention, and methods which use them,offer advantages over prior art cells, mammals and methods for studyingthe role of signaling pathway components which regulate oncogenesis,tumor growth, apoptosis and the development of drug resistance. In theprior art cells and methods, undefined tumors with relatively undefinedand unknown genotypes were used to study factors involved in regulatingapoptosis, chemosensitivity and the development of resistance totherapeutic agents. In contrast, the present invention provides modelcells and mammalian systems having a well-defined starting genotype.

In another embodiment, the invention provides a method for testing ahematopoietic tumor or tumor cell for sensitivity to a particular testtreatment. In certain aspects, the method involves administering a testtreatment to a mammal such as a mouse which harbors a population ofhematopoietic cells in which the activity of akt, bcl-2 and/or eIF4E isincreased or activated relative to a control animal and/or that of PTENand/or eIF3E is decreased or repressed relative to a control animal. Themouse has also been engineered to develop a hematopoietic tumor, whereinsome or all of the tumor cells have an activated signaling pathwaycharacteristic of increased akt, bcl-2 and/or eIF4E expression and/ordecreased PTEN and/or eIF3E expression. The mouse is monitored for thetime to onset of primary tumor formation (e.g., palpable tumorformation), and an increased time to tumor onset indicates sensitivityto the treatment. The invention thus further provides a method foridentifying agents or treatments which increase the time to tumor onsetin a mammal having a tumor or tumor cells with an activated signalingpathway characteristic of increased akt, bcl-2 and/or eIF4E expressionand/or decreased PTEN and/or eIF3E expression.

The instant invention is based in part on the discovery thathematopoietic tumors of a particular genotype may become resistant totreatments to which they were originally sensitive. Chemoresistance isassociated with a change in cellular signaling pathways involved inapoptosis (programmed cell death), and in particular, in akt, bcl-2 andeIF4E-mediated signaling.

Thus, in another embodiment, a mammal such as the above described mouse(i.e., having one or more hematopoietic tumors characterized by cellshaving increased akt, bcl-2 and/or eIF4E activity or reduced PTEN oreIF3E activity) is treated with an agent (e.g., chemotherapy) whichcauses remission of hematopoietic tumor cells. Those mice are thenmonitored for the length of time until (secondary) tumor relapse. Anincreased time of remission before tumor relapse indicates sensitivityof secondary tumors in the mice to the administered treatment. Theinvention thus further provides a method for identifying agents ortreatments which increase the time to tumor relapse in a mammal having atumor or tumor cells with an activated signaling pathway characteristicof increased akt, bcl-2 and/or eIF4E expression and/or decreased PTENand/or eIF3E expression, the mammal having been administered achemotherapeutic agent or treatment.

A lymphoma arising in a mouse that has received cells from Eμ-mychematopoietic stem cells transduced with a pMSCV-Akt vector can betested for sensitivity to a treatment, by administering the treatment tothe mouse (designated a “test” mouse), and monitoring the mouse for adecrease in signs of the lymphoma (remission). Complete remission is anormal state of the mouse in which no lymphoma can be detected bypalpation, and white blood cell counts are indistinguishable (notstatistically significantly different) from those of healthy mice.Sensitivity of a lymphoma to a treatment can be measured by the lengthof time until relapse, that is, the time when palpable tumors are againdetectable, or by the proportion of mice that survive a chosen period oftime. An appropriate control mouse when comparing the effects oftreatments, is for example, one which is genetically identical or verysimilar, and which is maintained under the same conditions as the testmouse, except that no treatment, or sham-treatment, is given.

The treatment to be tested can be one or more substances, for example, aknown anti-cancer agent, such as adriamycin, rapamycin, cylophosphamide,prednisone, vincristine, cisplatin, or a radioactive source. Thesubstance or agent can be administered preferably by intraperitonealinjection in a pharmaceutically acceptable vehicle, but also by otherappropriate vehicles and routes, for example, orally, intranasally, byinhalation, intramuscular injection, hypodermic injection, intravenousinjection or by surgical implantation, in topical creams, transdermalpatches and the like, all optionally with pharmaceutically compatiblecarriers and solvents. The treatment can also be exposure to variouskinds of energy or particles, such as gamma-irradiation, or can be acombination of approaches. In some cases, the treatment can also beadministration of one or more substances or exposure to conditions, or acombination of both, wherein the effects of the treatment as anti-cancertherapy are unknown.

Preferably, mammals, e.g., mice, used in the methods of the inventioncomprise a population of cells engineered to become hematopoietic tumorcells such as lymphomas and/or leukemic cells. In a preferredembodiment, the mammal expresses an oncogene such as myc from a strongviral promoter and/or enhancer, the overexpression of which results inthe development of hematopoietic tumorigenic cells and/or tumors. Apreferred mammal is a mouse harboring the Eμ-myc gene, which comprises amyc gene operably linked to an Eμ IgH enhancer (supra; see, e.g., Adamset. al., Nature 318:533-538 (1985)). An overexpressed myc gene, such asthe Eμ-myc gene, may be introduced into the cells of a mammal resultingin a transgenic mammal having the propensity to develop hematopoietictumors (see, e.g., U.S. Pat. No. 6,583,333). Alternatively or inaddition, an overexpressed myc gene, such as the Eμ-myc gene, may beintroduced into the cells of a mammal using one of the vectors of theinvention which further comprises a wildtype, variant, derivative ormutant form of a nucleic acid encoding a modulator or akt-, bcl-2,eIF4E, PTEN or eIF3E activity and which can infect or transduce a stemcell of the mammal that will develop into cells of the hematopoieticlineage. More preferably, the stem cell is a hematopoietic stem cell(pluripotent or multipotent). In another preferred embodiment, themammal comprise tumorigenic cells comprising a vector of the inventionbut has non-transgenic stem cells (e.g., produced by transplanting atransgenic tumor cell of the invention into a host syngeneic orsub-lethally irradiated mammal).

In certain preferred embodiments, activated myc in combination with akt,bcl-2 and/or eIF4E activities are introduced, individually or in anycombination, into stem cells or tumorigenic cells of the mammal by meansof a viral vector. The mammal, usually a mouse, readily develops primaryhematopoietic tumors of a uniform genotype, i.e., Eμ-myc in combinationwith one or more of the activated akt, bcl-2, eIF4E or repressed PTEN oreIF3E genes. Such mice are useful research tools for identifying genes,mutations, genetic alterations, treatments, agents and conditions whichalter the expression of these and other genes and activities involved incell growth, chemosensitivity through apoptotic signaling pathways, andthe development of resistance to such treatments through geneticalterations that affect those signaling pathways.

In another embodiment, a test treatment may be administered in vitro tohematopoietic tumor cells, such as lymphoma cells, that originally arosein a mouse engineered to develop a hematopoietic tumor. The tumor cellsexpress activated akt, bcl-2 or eIF4E activity or repressed PTEN oreIF3E activity (or any combination thereof). Treated tumor cells aremonitored for growth, wherein slowing or arresting of growth indicatessensitivity of the cell to the test treatment. As before, in preferredembodiments, the hematopoietic tumor cells also express activated mycfrom a strong viral promoter/enhancer such as an Eμ IgH enhancer.

Thus another embodiment of the invention provides a method for testing ahematopoietic tumor cell such as a lymphoma for sensitivity to atreatment, wherein the lymphoma arose in a mouse that received cellsfrom Eμ-myc hematopoietic stem cells transduced with a pMSCV-Akt vectorfor sensitivity to a treatment. Lymphoma cells are cultured in vitro, atreatment is administered to the cells (e.g., a drug is contacted withthe cells), and the cells can be monitored for growth (e.g., byobserving cell number, confluence in flasks, staining to distinguishviable from nonviable cells). A failure to increase in viable cellnumber, a slower rate of increase in cell number, or a decline in viablecell number, compared to cells which have been left untreated, or whichhave been mock-treated, is an indication of sensitivity to thetreatment.

In certain preferred embodiments, activated akt, bcl-2 and/or eIF4Eand/or repressed PTEN and/or eIF3E activities are introduced,individually or in any combination, into stem cells or tumorigenic cellsof the mammal by means of a viral vector, preferably before the testtreatment. In certain preferred embodiments, the viral vector is aretroviral vector which is capable of infecting or transducing stemcells which produce hematopoietic cells, such as MMLV- and MSCV-derivedretroviral vectors. Lentiviral vectors which can transduce stem cellsthat produce hematopoietic cells are also preferred. Any of the abovedescribed vectors of the invention, as are those which are used in theExamples set forth herein, may be used in this and other methods of theinvention.

In other embodiments, the invention provides methods for identifying atreatment that increases sensitivity of a hematopoietic tumor cell to achemotherapeutic drug or agent. The methods involve administering a testtreatment to a mammal (e.g., a mouse) that has been engineered todevelop a hematopoietic tumor. The mammal also comprises a population ofhematopoietic cells in which one or more of akt, bcl-2 or eIF4E activityhas been activated, or PTEN or eIF3E activity has been repressed. Themammal is monitored for the development of hematopoietic tumors and theextent to which such tumors are present in the mammal after the testtreatment is compared to the extent to which such cells are present in acontrol mammal. If tumor onset occurs less frequently in the test mousethan in the control mammal, the treatment is one which increaseschemosensitivity in an akt-, bcl-2, or eIF4E-activated cell to the testtreatment. Likewise, if remission from the primary hematopoietic tumorsoccurs more frequently in the test mammal than in the control mammal,the treatment is one which increases chemosensitivity in an akt-, bcl-2,or eIF4E-activated tumor to the test treatment. In contrast, if tumoronset occurs more frequently in the test mammal than in the controlmammal, or if remission from the primary hematopoietic tumors occursless frequently in the test mammal than in the control mammal, thetreatment is one which decreases chemosensitivity in an akt-, bcl-2, oreIF4E-activated cell to the test treatment.

Thus a further embodiment of the invention provides a method employingsecondary tumors arising from transplanted primary tumor cells (e.g. seeExamples 2, 9-11). In this way, a statistically significant number ofmice with the same lymphoma can be studied for their response to aregimen of therapy. One or more primary tumors can be harvested from ananimal and transplanted into recipient mice by a suitable method, suchas by tail vein injection. After a period of time, secondary lymphomasarise in the recipient mice. A treatment is then administered to onepopulation of recipient mice which have developed tumors (making a“treated” population). A second population of recipient mice can serveas controls and remain untreated or be sham-treated. Thereafter, thetreated population of mice is monitored for remission (ordinarily, bypalpation for tumors). The proportion and length of remissions among thetreated population indicate the extent of sensitivity of the lymphomasto the administered treatment(s). If relapse occurs, and further tumorsarise, the process can be repeated. Lymphomas arising upon relapse canbe collected for the study of resistance to the therapy.

The invention further provides a method for detecting a geneticalteration in a cell, the alteration which is associated with resistanceto a treatment that normally causes remission of a tumor. Tumor cellsare harvested from a mammal (e.g., a mouse) that is or which haspreviously been engineered to develop hematopoietic tumor cells (e.g.,which overexpresses myc, as described above). The mammal is alsoengineered to have a population of hematopoietic cells (e.g., bytransducing stem cells or primary tumor cells) characterized by anactivated akt, bcl-2 or eIF4E activity (or any combination thereof),and/or repressed PTEN and/or eIF3E activities. Tumor cells aretransplanted into recipient mice and tumors permitted to arise. Therecipient mice are then treated with a therapeutic agent, therebyachieving remission, and the recipient mice are monitored for relapsefollowing remission. Secondary tumor cells are isolated from recipientmice. In some embodiment, tumor cells are optionally passaged throughdifferent recipient mice, one or more times. A measurable difference inthe level or function of a gene or gene product between the tumor cellsof the last performed step and primary tumor cells of the first step isindicative of a genetic alteration in the subsequently formed tumors.

The step of identifying a difference in the level or function of a geneproduct may be performed by any of numerous methods well known in theart, preferably using high throughput methods and arrays or microarrays.Such methods may be designed to monitor RNA expression (e.g., by nucleicacid hybridization and other such techniques). In addition, methodswhich measure protein expression may also be used. As described above,in preferred embodiments, tumor cells of a well-defined genotype areused in the methods. Preferably, the tumor cells express activated myc,and one or more of the following: activated akt, bcl-2, eIF4E orrepressed PTEN or eIF3E activities.

In preferred embodiments, stem cells are engineered by introduction ofan activated akt-, bcl-2, and/or eIF4E (alone or in combination), and/ora repressed PTEN and/or eIF3E. One or more of these activities may beintroduced into a stem cell which expresses activated myc (or anotheroncogenic activity) which results in formation of hematopoietic tumorsin a mammal. Alternatively (but not mutually exclusive), activated mycmay be introduced in conjunction with one or more of the activated akt-,bcl-2, and/or eIF4E (alone or in combination), and/or a repressed PTENand/or eIF3E functions on a viral vector, such as a retroviral vector,as described above. Any of the vectors of the invention, and cellstransduced with them, may be useful in such methods. Preferably, thestem cells are human cells, and more preferably, are hematopoietic stemcells. In certain embodiments, primary tumor cells are transplanted intonew recipient mice and tumors are allowed to form. A treatment is thenadministered to recipient mice, thereby achieving tumor remission. Miceare monitored for the appearance of tumors (relapse) and secondary tumorcells are harvested from recipient mice. The steps of transplanting,treating to achieve remission and monitoring secondary tumor formationare optionally repeated in sequence one or more times, using differentrecipient mice with each repetition.

Methods for obtaining hematopoietic tumor cells for the study of drugresistance associated with a test gene (or genetic alteration) are alsoprovided. In one such method, a treatment is administered to miceengineered to form hematopoietic tumorigenic or tumor cells (e.g., micecomprising a population of cells having a genome comprising a myc geneoperably linked to an Eμ IgH enhancer); and further comprising the testgene by insertion into the genome of a retroviral vector (e.g., pMSCV)bearing the gene. Such mice develop a hematopoietic tumor or apre-malignant condition due to the activated myc gene, and the treatmentcauses remission of the tumors or pre-malignant cells in the mice. Themice are monitored for tumor relapse and steps are optionally repeatedin sequence one or more times. Hematopoietic tumor cells, such aslymphoma cells, are then harvested from the mice of the last performedstep. Harvested cells are useful, e.g., in methods of the inventioninvolving screening for genes and therapeutic agents which affecttumorigenesis and apoptosis-related pathways involved in the control ofchemoresistance in tumor cells, especially hematopoietic tumor cells.One advantage of such isolated tumor cells is that they are of arelatively well-defined or known genotype, as the mammalian model is onewhich is engineered to develop hematopoietic tumors (e.g., byoverexpression of an oncogene such as myc), and has an activated akt,bcl-2 and/or eIF4E activity and/or a repressed PTEN and/or eIF3Eactivity to create chemoresistance in the model.

In another embodiment, the invention provides methods for testing theeffects of a treatment on tumor growth by transducing (Eμ-myc)hematopoietic stem cells with a vector comprising an activated akt,bcl-2, and/or eIF4E activity (or repressed or mutated PTEN or eIF3Eactivity); administering such transduced cells to irradiated recipientmammals, wherein the recipient mammals develop primary tumors;harvesting a primary tumor from an irradiated animal; administeringcells derived from the primary tumor to a recipient animal;administering a treatment to the recipient animal; and monitoring therecipient animal for the effect of the treatment on tumor growth. Inthis embodiment, if tumor growth is decreased or increased in therecipient animals relative to tumor growth in control animals, there isan inhibitory or enhancing effect, respectively, of the treatment ontumor growth. Preferred embodiments of this method parallel thepreferred embodiments of methods described above, insofar as any vectoror cell of the invention may be used. In one preferred embodiment, thetreatment comprise an agent that is a member of a chemical library.

The invention further provides a method for testing a nucleic acid forits effect on tumorigenesis and/or sensitivity of a tumorigenic cell toa treatment. The method involves lethally irradiating a recipientmammal; introducing a test gene into stem cells engineered to producehematopoietic tumors in the recipient mammal; reconstitutinghematopoietic cells of the recipient mammal with such altered stemcells; and observing the effect of the test gene on tumorigenesis in therecipient mammal. Preferably, the stem cells are engineered to producehematopoietic tumors in a mammal by means of an activated myc gene. Inanother preferred embodiment, a primary tumor cell (rather than a stemcell) is transduced with the test gene and introduced into a syngeneicand/or sub-lethally irradiated mammal. In certain preferred embodiments,the test gene is introduced into a stem cell or tumor cell before or incombination with a nucleic acid encoding activated myc. Thus, thestarting stem cell or tumor cell may but need not be a cell geneticallyengineered to overexpress myc. In certain preferred embodiments, thestem cell or tumor cell is derived from a patient and the methods of theinvention used to diagnose which therapy combination will be mosteffective in preventing or diminishing tumor onset and/or in preventingthe development of chemoresistance in an akt-, bcl-2-, eIF4E-activatedhematopoietic tumor cell.

In a more preferred embodiment, a nucleic acid encoding an activatedakt, bcl-2 or eIF4E activity, and/or a repressed PTEN and/or eIF3Eactivity, is also introduced into the stem cell or tumor cell, alone orin any combination. One of more of these activities may be introducedinto a myc-expressing cell, or myc activity may be introduced inconjunction with activation of the akt-, bcl-2, eIF4E signaling pathway.Further, the test gene may be administered together or separately fromthe component(s) of the akt-, bcl-2, eIF4E signaling pathway. In anotherpreferred embodiment, the test gene is introduced into stem cells ortumorigenic cells as a member of a collection of nucleic acid moleculescontained in a library. The library can be any of a number of nucleicacid libraries, including cDNA or synthetic expression libraries, shRNA(RNAi) inhibitory libraries and the like.

A further embodiment of the invention is a test for the effect of a geneon sensitivity of a lymphoma to a treatment (e.g., anti-cancer drug). Inone embodiment, cells can be produced from a lymphoma described herein.A gene to be tested for its effect on the sensitivity to a treatment ofthe lymphoma is introduced into the cells, such that the cells producethe gene product.

Introduction of the gene can be, for instance, by transformation, suchas by electroporation, by calcium phosphate, DEAE-dextran, or byliposomes, using a vector which has been constructed to have aninsertion of the gene. See chapter 9 in Ausubel, F. M. et al, CurrentProtocols in Molecular Biology, containing supplements throughSupplement 63, (2003), John Wiley & Sons, New York.

The introduction of a gene of interest can also be accomplished by viralinfection, for example, by a retrovirus. Retroviral gene transfer hasbeen used successfully to introduce genes into whole cell populations,thereby eliminating problems associated with clonal variation(McCurrach, M. E. et al., Proc. Natl. Acad. Sci. USA 94:2345-2349,(1997); Samuelson, A. V. and Lowe, S. W., Proc. Natl. Acad. Sci.USA:12094-12099, (1997); Serrano, M. et al., Cell 85:27037, (1997)).

Tumor cells so altered or transformed by the introduced gene, orunaltered cells, (“unaltered” including cells which have beentransformed with a control vector, transfected with control DNA, orinfected with a control virus (control constructs not carrying the geneof interest), can be introduced into immunocompetent recipient mice(“test” mice receiving the gene and “control” mice not receiving thegene). Cells can be introduced by injection, for example, by injectioninto the tail vein of the mice. Lymphomas are allowed to form in boththe test and control mice, and both test and control mice are monitoredfor the development of lymphomas (for example, by palpation for tumors,or by detection of green fluorescent protein in tumors geneticallyaltered to produce GFP). Both groups of mice are administered atreatment, preferably a drug given at a dose with a known anti-tumoreffect, and monitored for remission. A difference in the frequency ofremissions in the test mice compared to that of the control mice(remissions are not anticipated in control mice), indicated by anincrease in the percentage of tumor cells expressing the gene in treatedcompared to untreated mice), indicates an effect of the gene on theresponse of the lymphoma to the treatment. This method can be used tolook for test genes which increase or decrease the effectiveness of atest treatment on tumor formation.

This method can determine what the response of an animal would be to thesame therapy, with and without the gene. Thus, genes that are importantto drug sensitivity of a lymphoma, or analogously, to another tumor celltype, can be identified.

In yet another preferred embodiment, the invention provides a method forinhibiting growth of an akt-, bcl-2-, and/or eIF4E-activated tumor cellin a mammal, by administering an effective dose of rapamycin or activeanalog thereof in combination with a chemotherapeutic agent. Thechemotherapeutic agent is preferably one (or more) selected from thegroup consisting of adriamycin, cyclophosphamide and doxorubicin.

In another preferred embodiment, the invention provides a method forassessing the sensitivity of a tumor to a test chemotherapeutic agent.The method involves determining whether the tumor expresses activatedakt, bcl-2 or eIF4E activity; and treating such an activated tumor withrapamycin or an active analog thereof in combination with the testchemotherapeutic agent. The tumor is sensitive to the testchemotherapeutic agent if regression or remission occurs following thetreatment in comparison to control treatments. Preferably, the testchemotherapeutic agent is a DNA modifying agent such as one of a numberof DNA damaging agents, DNA cross-linking agents, DNA alkylating agentsand topoisomerase inhibitors that cause DNA damage. In more preferredembodiments, the agent is cyclophosphamide, melphalan, doxorubicin ordaunorubicin. In other preferred embodiments, the test chemotherapeuticagent is expressed as a member of a nucleic acid or a chemical library.

In yet another aspect, the invention provides a method for inhibitingthe growth of akt, bcl-2 or eIF4E-activated tumors in a mammal,comprising administering to the mammal an effective dose of an inhibitorof translation in combination with an effective dose of one or morechemotherapeutic agents. Preferred inhibitors of translation include butare not limited to: 4E-BP(1, 2 or 3); tsc-1 and tsc-2; the N-terminaldomain or a dominant-negative mutant of eIF4G (Morino et al., Mol CellBiol. 20(2):468-77 (2000)); a dominant-negative mutant of eIF4A (Oguroet al., RNA 9(4):394-407 (2003); Pdcd4 (Yang et al., Mol Cell Biol.23(1):26-37 (2003); picornavirus protease 2A (Haghighat et al., J.Virol. 70(12):8444-50 (1996); and NSP3 (Padilla-Noriega et al., Virology298(1):1-7 (2002)).

In other aspects, the invention provides a method for identifying apotential target for drug therapy in the treatment of an akt, bcl-2 oreIF4E-activated tumor, the method comprising harvesting hematopoietictumor cells from a mouse engineered to develop a hematopoietic tumor andbearing a population of cells comprising an activated akt, bcl-2 oreIF4E activity (or repressed PTEN or eIF3E activity); and introducing anucleic acid library into a population of the harvested cells. Afraction of cells comprising one or more members of the nucleic acidlibrary is transplanted into recipient mice; and hematopoietic tumorcells allowed to arise in transplanted recipients. Earlier recurrenceand reduced survival of a recipient mouse compared to control miceindicates the presence of a potential target encoded by at least onemember of the introduced nucleic acid library. The potential target maybe characterized further by well known methods, including DNAsequencing, genetic analyses, expression array technology (at thenucleic acid and/or protein level); two-hybrid screening and the like.

The invention further provides a method for identifying a drug fortreatment of an akt, bcl-2 or eIF4E-activated tumor, comprisingharvesting hematopoietic tumor cells from a mouse engineered to developa hematopoietic tumor and bearing a population of cells comprising anactivated akt, bcl-2 or eIF4E activity (or repressed PTEN or eIF3Eactivity); making extracts of such hematopoietic tumor cells andtreating them with one or more test molecules. The effects of the testtreatment on one or more markers of the akt-, bcl-2, eIF4E signalingpathway are assessed by conventional means. A change in the phenotype ofa marker of the akt signaling pathway in the direction of decreased akt,bcl-2 and/or eIF4E activity indicates the presence of a potential drug.Likewise, a change in the phenotype of a marker of the akt signalingpathway in the direction of increased PTEN activity indicates thepresence of a potential drug. Markers of the akt signaling pathwayinclude akt, p-akt, TSC1/2, p-TSC1/2, Rheb, p-Rheb, mTOR, p-mTOR, s6kinase, p-s6 kinase, S6, p-S6, 4E-BP and p-4E-BP, eIF4G, p-eIF4G, eIF4B,p-eIF4B, bcl-2, p-bcl-2, FKHR, p-FKHR, bad, p-bad, GSK, p-GSK, eIF2B,p-eIF2B. Markers of the akt signaling pathway also include caspase-9phosphorylation, bad cytoplasmic localization, mdm-2 phosphorylation andcytoplasmic localization, p27 cytoplasmic localization, forkheadtranscription factors cytoplasmic localization, changes in glucosemetabolism and reduced apoptosis.

Conventional methods may be used to assess the presence of such markersin test- and control-treated animals or cell extracts. Such methodsinclude but are not limited to immunoblotting, immunofluorescence,immunohistochemical staining, co-immunoprecipitation with cytoplasmicproteins (e.g. 14-3-3 proteins); functional assays for metabolic markers(e.g., high glucose uptake by PET, etc.), apoptosis, reduced p53induction following DNA damage/stress, measures of RNA levels such asNorthern blot analysis, RT-PCR (e.g., quantitative, Real-Time [RT] PCR).

The invention described herein exploits mouse models to generate tumorswith specific genetic alterations, allowing the production of usefultools for future drug discovery programs applicable to human lymphomas.By understanding the regulation and execution of apoptosis in tumorcells, it may be possible to selectively increase the chemosensitivityof tumor cells, or to develop novel therapies to activate apoptosis moredirectly. The results of terminal deoxynucleotidyl transferase-mediateddioxygenin-11-dUTP nick end labeling (TUNEL) and Ki67 analyses ofhistological sections from Eμ-myc/Akt and Eμ-myc/Bcl-2 tumors (seeExamples herein) suggest that Akt, like Bcl-2, interferes withapoptosis.

The approach of introducing the Akt oncogene into hematopoietic stemcells by retroviral vector creates a valid and relevant model for the invivo evaluation of agents targeting the PI3K/Akt pathway. Specifically,it can be useful in target validation, selection of potent inhibitors ina physiological setting, and the design of therapeutic combinationregimens most active in cancers that display high levels of Aktactivity. For example, the approach can be used to test whetherrapamycin—an inhibitor of the Akt target mTOR—will be effective againstthe Akt-overexpressing lymphomas, or reverse their profound resistanceto conventional agents. The approach is more broadly applicable to othergenes, for example, genes suspected of encoding gene products with arole in tumor maintenance, or a role in apoptosis as effectors,inhibitors, or the like.

Lymphoma cells—primary or secondary—generated by the methods describedherein can be studied for differences in the level of gene copy numberor gene expression by a number of methods known in the art. For exampleanalysis of polymerase chain reaction (PCR) products using lymphoma cellDNA as template, and primers to amplify genes of interest. PCR can becombined with capillary electrophoresis (CE), as in integrated PCR-CE ona microchip. Recent technology enables one to detect variation in genomecopy number by identifying small deletions or amplifications of genesunder various treatment conditions (see Lucito R., et al.,“Representational oligonucleotide microarray analysis: a high-resolutionmethod to detect genome copy number variation”, Genome Res.,13(10):2291-305-(2003)). One may also use genomic arrays such as CGH orROMA to analyze gene copy number. DNA microarrays can be used toidentify gene expression patterns and to detect differences in suchpatterns, for example, with the aid of software designed for thepurposes of such comparisons. Differences in gene function can beanalyzed by RNA interference, for example, as in Mousses S. et al.,Genome Res. 13(10):2341-2347 (2003). Other methods to study differencesin gene expression resulting from mutations in the lymphoma cells arethose that analyze the proteins produced by the cells. Proteins producedin cells can be separated and identified by protein profiling, e.g., by2-D gel electrophoresis and mass spectrometry. Protein arrays can beproduced, and proteins can be characterized and identified, using, forexample, matrices of peptides, antibodies, apatamers, or chromatographicsurfaces. Profiles of the DNA, RNA or proteins produced by such methodscan be compared between primary lymphomas sensitive to a treatment andsecondary lymphomas that have acquired one or more mutations to becomeresistant to the treatment.

Our results provide new insights into Akt signaling and tumor behavior.We find that Akt lymphomas are phenotypically similar to those harboringa purely anti-apoptotic oncogene with respect to their acceleratedonset, aggressive pathology, ability to retain p53 and chemotherapyresistance. Thus, despite its pleiotropic activities, the pro-survivalfunctions of Akt appear sufficient to promote tumorigenesis. We furthershow that the mTOR inhibitor rapamycin can restore apoptotic sensitivityto lymphomas expressing Akt, and that the translation factor eIF4E canrecapitulate Akt action in vivo. Consequently, a substantial proportionof Akt survival signaling may result from deregulated translation,perhaps through altering the recruitment of pro- and anti-apoptoticmRNAs to polysomes (Grolleau, A., et al., “Global and specifictranslational control by rapamycin in T cells uncovered by microarraysand proteomics,” J. Biol. Chem. 277: 22175-22184 (2002) and Rajasekhar,V. K., et al., “Oncogenic Ras and Akt signaling contribute toglioblastoma formation by differential recruitment of existing mRNAs topolysomes,” Mol. Cell. 12: 889-901 (2003)). Of note, the apparent roleof translational control in cell survival extends to tumors with otherPI3K pathway lesions, since the chemotherapy resistance ofPTEN-deficient lymphomas is reversed by rapamycin in a manner that isblocked by eIF4E. Together, these results provide in vivo evidence thattranslational signaling through Akt is a broadly relevant oncogenesisand drug resistance mechanism. They also provide the first evidence thatoverexpression of eIF4E promotes oncogenesis and drug resistance.

The invention further provides a means for directly comparing eIF4E toAkt in the Eμ-myc model, in order to further examine the effects oftranslational control on tumor phenotypes. In one preferred embodiment,the encoded eIF4E polypeptide has altered biological activity. Incertain embodiments, it is preferred that eIF4E have enhanced oroverexpressed activity. Example 11, for example, shows how hematopoieticreconstitution with Eμ-myc transgenic HSCs expressing eIF4E acceleratelymphomagenesis in a manner highly similar to Akt overexpression.Moreover, the invention further provides a method for testing thechemosensitivity of eIF4E tumor cells by quantifying the disseminatedpathology and proliferation/apoptosis ratio of the lymphomas.

In another embodiment, the slowing or arresting of tumor growth in micehaving one or more hematopoietic tumors characterized by cells havingaltered (i.e. increased) eIF4E activity can be tested for byadministering a drug treatment. The said treatment comprises achemotherapeutic agent which causes remission of hematopoietic tumorcells. The time to tumor relapse or remission can be used as a measureof chemoresistance or chemosensitivity. Example 11 provides data showingthat the eIF4E transgene confers a high degree of resistance to variouschemotherapeutic treatments, including rapamycin and doxorubicin.

The invention further describes a mouse model to generate tumors withspecific genetic alterations, wherein the mice show a mutated (i.e.altered; increased or decreased) expression of PTEN or eIF4E relative towild-type control mice. In a preferred embodiment, Eμ-myc/PTEN+/− miceare derived from a cross of a transgenic Eμ-myc mouse to a transgenicPTEN+/− mouse and the method includes detection of a loss ofheterozygosity in the PTEN locus by allele specific PCR. Example 12illustrates an enhanced susceptibility to tumors in the PTENheterozygous mice, and further characterizes an enhanced chemoresistancein these mice in vivo. PTEN heterozygosity thus causes a significantacceleration and increased penetration in lymphoma development in vivo.The invention further describes a lymphoma development in Eμ-myc/PTEN+/−mice that is resistant to chemotherapy, namely doxorubicin, and reversedby rapamycin.

A further embodiment of the invention is a method for transducing HSCsinto recipient Eμ-myc/PTEN+/− mice wherein the HSCs are infected with aretroviral pMSCV-eIF4E vector, or with the particular pMSCV-eIF4Evector, pMSCV-eIF4E-IRES-GFP. A transduction procedure is outlined inExample 13. A preferred embodiment characterizes a method forquantifying changes in chemoresistance in eIF4E infected tumor cellsderived from said Eμ-myc/PTEN+/− mice, wherein eIF4E confers resistanceto Eμ-myc/PTEN+/− tumors after in vivo drug treatment (see Example 13).

The invention further provides a method for harvesting Eμ-myc HSCs andinfecting them in vitro with a pMSCV-eIF4E vector, or with theparticular pMSCV-eIF4E-IRES-GFP vector expressing different mutants ofeIF4E. A preferred embodiment includes mutants of various residues ofproteins that are important in the Akt pathway and more preferably thatare important in the function of eIF4E. Example 14 shows that mutatingsome of these proteins, namely E103A, S209A, and W56A, can affecteIF4E's oncogenicity in vitro. In particular, S209 induces tumors atlower and much delayed penetrance. This would indicate that thephosporylation site S209 is important for eIF4E's oncogenicity.Interestingly, the S209A mutant affects eIF4E function without affectingits interaction with 4E-BP. The invention thus provides a method ofmapping functional domains of a protein that play a role in oncogenesisand/or chemoresistance. As phosphorylation at this site is mediated viap38 and MEKK activation of MNK-1, this invention also indicates, thateIF4E tumors (and similarly Akt expressing tumors) should be sensitiveto pharmacological inhibitors of these kinases.

The invention further provides a method to investigate whether RNAiapplied to a well-characterized gene can stably suppress gene expressionin HSCs and produce detectable phenotypes in mice. A retroviral mediatedgene transfer is illustrated in Example 15. Herein Eμ-myc mice areinfected with a short hairpin against PTEN (shPTEN). Infection occurs bytransduction of cells with a pMSCV-shPTEN vector. A preferred embodimentis the use of a particular pMSCV-shPTEN-IRES-GFP vector. Two murine PTENsequences can be used, as described and disclosed in Example 15; PTENsh1 and PTEN sh2. Transduced cells integrate the pMSCV-shPTEN-IRES-GFPvector in the cell genome and induce lymphomas in the Eμ-myc mousemodel.

The invention also encompasses a method for testing the chemoresistanceof cells that have been transduced with pMSCV-shPTEN vectors. The timeto tumor development is shortened following reconstitution of lethallyirradiated mice infected with the pMSCV-shPTEN vectors. Moreover, PTENexpression is downregulated in the presence of shPTEN, as illustrated bya decreased expression of endogenous total and phosphorylated PTEN bywestern blotting in Example 15. The combination of vector, cells andmethods that allow the introduction of shPTEN RNAi into the system toknockdown PTEN and create activation of endogenous Akt is a furtherembodiment of the invention that enables a broader analysis of loss offunction changes in the Eμ-myc mouse model.

Accordingly, the present invention is also directed to modulating theactivity of translation factors in the treatment of diseases, conditionsand states associated with abnormal proliferative growth phenotypes,especially those which are lymphoproliferative or myeloproliferative.The invention provides compositions and methods for preventing Akt,Bcl-2 or eIF4E-mediated inhibition of apoptosis to increasechemosensitivity of tumor cells such as those expressing activated Aktsignaling components.

In one embodiment, the invention thus provides inhibitors of eIF4Eactivity in a cell. Also provided are cells in which eIF4E activity isinhibited, and mammals comprising such cells, particularly hematopoieticcells and more particularly, hematopoietic stem cells. The presentinvention also provides screening methods for identifying agents thatwill inhibit eIF4E activity, thereby deregulating the akt pathwaysignaling which affects apoptosis and chemosensitivity or resistance. Apreferred inhibitor of eIF4E activity is 4E-BP (binding protein), whichis a repressor of eIF4E activity. 4E-BP inhibits eIF4E activity bysequestering eIF4E in an inactive form. eIF4E thus shuttles between anunbound state or a state where it is bound to eIF4G (and participates intranslational initiation), and the 4E-BP bound, inactive state.Accordingly, overexpression of 4E-BP in a cell of the invention willmodulate apoptosis and decrease akt-/bcl-2-/eIF4E-mediatedchemoresistance in a tumor cell in vivo.

It is also envisioned that inhibiting other general translation factors(i.e., molecules which participate directly in initiating translation ofmRNA into protein) tumor cells such as hematopoietic tumor cells willhave the desired effect of increasing apoptosis and reducingchemoresistance. Examples of suitable targets for inhibition include thetranslation factors eIF3 (subunits thereof), eIF1, eIF5, eIF2 (subunitsthereof), eIF2B, eIF1A, eIF4A (isoforms I, II and III), eIF4B, eIF4G(isoforms I and II), eIF4H, eIF4F (which is a complex of eIF4E, 4A, and4G), eIF5A, eIF5B, and elongation factors eEF1A1, eEF1A2, eEF2, as wellas the eIF4E related protein, eIF4E-HP, and the eIF4E transporterprotein, eIF4E-T. Also included are the poly A Binding Protein (PABP)and translational termiantion factors such as eRF1 and eRF3 (eukaryoticrelease factors 1 and 3). See, e.g., Table 1. TABLE 1 Eukaryotic proteinsynthesis inhibitors In Vivo In Vitro Inhibitor Site of Inhibitions^(a)Inhibition^(b) Inhibition Arginine- Elongation − + AminoglycosideConjugates 4-Aminohexose Transpeptidation, + + Pyrimidine NucleosidesAgonist of Stress-Activated Protein Kinase AnisomycinTranspeptidation, + + agonist of Stress-Activated Protein KinaseAurintricarboxylic acid Ternary Complex − + Formation Baciphelacin NotKnown + + Bouvardin EF-2 dependent + + translocation ClotrimazoleeIF-2a + n.t. phosphorlylation, Depletion of Ca++ stores CryptopleurineTranspeptidation + + Didemnin eEF-1α + + Diterpene Esters Peptidyltransferase + + (Genkwadaphnin, yuanhuacine) Edeine Initiation and ?? +elongation by interfering with P site function Eicosapentaenoic AcidPhosphorylation of + − eIF2a Emetine Family Translocation + + FlavinoidsPhosphorylation of + n.t. eIF2a Fusidic acid Translocation − +Glutarimide Family Translocation + + (e.g.-cycloheximide) HarringtonineFamily Met-tRNAi binding + + 2-(4-methyl-2,6- Initiation + + dinitro-anilino)-N-methyl- propionamide (MDMP) M⁷GDP Initiation − + NagilactoneElongation + + Negamycin Initiation, miscoding, + + termination^(a)Refers to the specific partial reaction inhibited or otherwiseaffected.^(b)n.t.—not reported or not known.

Our results also have profound implications for considering the use oftargeted therapeutics alone or in combination with conventionalchemotherapeutic agents. The result that rapamycin can reverse drugresistance in Akt-expressing tumors is compelling evidence thatreversing apoptotic defects can restore drug sensitivity in vivo.Furthermore, the ability of eIF4E to mediate drug resistance downstreamof Akt implies that inhibitors of translation initiation may also beeffective chemosensitizing agents, and perhaps less prone to resistance.The selectivity of rapamycin to reverse drug resistance in Aktexpressing lymphomas—despite their broad similarities to those withother apoptotic lesions—implies that the utility of this combinationtreatment would be missed in clinical trials based solely on tumor typeor pathology. Together, these results provide in vivo validation for astrategy to reverse drug resistance in human cancers, and underscore theimportance of tailoring cancer therapy based on tumor genotype.

The invention further provides methods for treating a disease, conditionor disorder associated with abnormal regulation of cell proliferation,especially abnormal lymphoproliferative or myeloproliferative states.Diseases and disorders include but are not limited to: Non-Hodgkin'slymphoma (NHL) and Hodgkin's lymphoma; follicular lymphoma (FL); smallcell lymphoma, diffuse large cell lymphoma; Burkitt's lymphoma; B cellchronic lymphocytic leukemia (B-CLL); T cell lymphocytic leukemia(T-CLL); B cell acute lymphocytic leukemia (B-ALL); T cell acutelymphocytic leukemia (T-ALL); chronic myelogenous leukemia (CML);myelodysplastic syndrome (MDS); promyelocytic leukemia (PML); acutemyelogenous leukemia (AML); autoimmune-lymphoproliferative syndrome(ALPS); multiple myeloma (MM); and plasmacytoma.

The following are examples which illustrate the compositions and methodsof this invention. These examples should not be construed as limiting:the examples are included for the purposes of illustration only.

EXAMPLES

Methods:

Isolation of Tumor Cells and Genotyping PCR

Lymphoma-bearing E-myc transgenic mice (C57BL/6 inbred strain) weresacrificed by CO₂ euthanasia. Dissected lymph nodes were minced inphosphate buffered saline (PBS) and filtered the material through a 35-mnylon mesh. The tumor cells were sorted for green fluorescent protein(GFP) content by flow cytometry (FACScalibur, Beckton Dickinson). DNAwas isolated from the purified GFP positive population by standardprocedure and loss of the remaining wild-type allele [loss ofheterozygosity (LOH)] of p53 or PTEN in tumors originating from p53 orPTEN heterozygous hematopoietic stem cells (HSC), respectively, wasdetected by allele-specific PCR as described (Schmitt, C. A. et al.,Genes Dev 13(20):2670-2677, (1999); Di Cristofano A., et al., NatureGenetics, 19(4):348-355, (1998)).

In Vivo Chemotherapy

Tumor cells were transplanted from the Akt-expressing tumors and fromcontrol tumors (Eμ-myc, arf^(−/−), p53^(+/+)) into syngeneic,non-transgenic 6-10 week old C57BL/6 mice by tail vein injection [10⁶viable lymphoma cells in phosphate-buffered saline (PBS)] and recipientmice were monitored for tumor formation twice a week by palpation of thepre-scapular and cervical lymph-nodes. When tumors became well palpable,adriamycin (ADR) or cyclophosphamide (CTX) was given at a single dose of10 mg/kg or 300 mg/kg body weight intraperitoneally (i.p.),respectively. Following treatments, mice were monitored for immediateresponse and time to relapse by lymph node palpation and by blood smears(by tail artery bleeding) stained according to a modified Wright'sprotocol using the Leukostat kit (Fisher Scientific, Pittsburgh, Pa.).The data were statistically evaluated.

A ‘complete remission’ (CR) was defined as absence of any palpable tumorand the absence of leukemia, as examined by blood smears. ‘Tumor freesurvival’ was defined as the time between treatment and reappearance ofa well-palpable lymphoma (Schmitt, C. A., et al., “Genetic analysis ofchemoresistance in primary murine lymphomas,” Nat. Med. 6: 1029-1035(2000)). ‘Overall survival’ was defined as the time between treatmentand progression to a terminal stage at which the animals weresacrificed. Tumor free and overall survival data were analyzed in theKaplan-Meier format using the log-rank (Mantel-Cox) test for statisticalsignificance. Whole body fluorescence imaging of living animals wasperformed as described (Schmitt, C. A., et al., “Dissecting p53 tumorsuppressor functions in vivo,” Cancer Cell 1: 289-298 (2002) and Yang,M., et al., “Whole-body optical imaging of green fluorescentprotein-expressing tumors and metastases.” Proc. Natl. Acad. Sci. USA97: 1206-1211 (2000)).

Rapamycin (RAP) was given at 4 mg/kg, i.p.×5 d, doxorubicin (DXR) at 10mg/kg, i.p., cyclophosphamide (CTX) at 300 mg/kg, i.p., or variouscombinations of drugs were given. In combination studies, the cytotoxicagent was given on day 2 of the rapamycin protocol. Rapamycin (LC labs,MA) was initially dissolved at 10 mg/ml in 100% ethanol, stored at −20°C., and further diluted in an aqueous solution of 5.2% TWEEN 80 and 5.2%PEG 400 (final ethanol concentration, 2%) immediately prior to use.Doxorubicin (Sigma) and cyclophosphamide (Sigma) were dissolved insaline. In treatment studies, control lymphomas were ARF-null tumorsarising in an Eμ-myc/ARF−/− background (Schmitt, C. A., et al.,“INK4a/ARF mutations accelerate lymphomagenesis and promotechemoresistance by disabling p53,” Genes Dev. 13: 2670-2677 (1999)).

Histopathology

Tissue samples were fixed in 10% buffered formalin and embedded inparaffin. Thin sections (5 μm) were stained with hematoxylin and eosinaccording to standard protocols. Detection of phosphorylated Akt [rabbitantibody against Hematopoietic stem cell-Akt Ser 473, 1:100 (CellSignaling Technology)] and Ki-67 [rabbit antibody, 1:100 (NovoCastra)]was by standard avidin-biotin immunoperoxidase method, usingbiotinylated goat or rabbit specific immunoglobulins (Vector labs) at1:500 and avidin-biotin peroxydase complexes (1:25, Vector Labs).Diaminobenzidine was used as the chromogen and hematoxylin ascounterstain. For B220 immunohistochemistry [rat antibody against mouseCD45R/B220-clone RA3-6B2 (BD Biosciences, Pharmingen)], antigenretrieval was required and a biotinylated antibody against rat was usedas a secondary antibody. The apoptotic rate was analyzed by TUNEL assayaccording to published protocols (Di Cristofano, et al., “Pten andhematopoietic stem cell27KIP1 cooperate in prostate cancer tumorsuppression in the mouse,” Nat. Genet. 27: 222-224 (2001)).

Western Blotting Analysis

Whole-cell lymphoma cell lysates were generated by lysing and extractingcells in SDS sample buffer (60 mM Tris-HCl at pH 6.8, 10% glycerol, 2%SDS, and 5% 2-mercaptoethanol). Samples corresponding to 50 μg ofprotein (Bio-Rad Bradford protein assay) were separated on anSDS-polyacrylamide gel and transferred to Immobilon-Hematopoietic stemcell membranes (Millipore). Total and phosphorylated Akt and total andphosphorylated S6 kinase were detected using antibodies 9272, 9275, 9202and 9206 respectively, (all from Cell Signaling Technology, 1:1000dilution). Antibodies against ribosomal S6 protein (2212, 1:1000, CellSignaling), phospho-ribosomal S6 (2215, 1:1000 Cell Signaling), cleavednuclear poly ADP-ribose polymerase (PARP) (9548, 1:1000 Cell Signaling),Bcl-2 (N19, 1:200, Santa Cruz), eIF4E (9742, 1:2500, Cell Signaling),4E-BP1 (9452, 1:1000, Cell Signaling), and β-actin (1:5000, Sigma) werealso used as probes in western blot experiments. α-Tubulin, as loadingcontrol, was detected using the monoclonal antibody B-5-1-2 (1:5000,Sigma). Proteins were visualized using enhanced chemiluminescence (ECL,Amersham, and Lumilight, Roche) or Supersignal (Pierce). Quantificationof eIF4E expression was quantified by immunoblotting using the aboveeIF4E primary antibody and [I¹²⁵]-recombinant protein A (at 0.1 μCi/ml;specific activity of 70-100 μCi/μg; PerkinElmer, Ontario, Canada) as asecondary reagent. The blot was exposed to a phosphor imager plate andscanned on a Fuji film BAS 1800II machine.

Lymphoma Cell Culture and In Vitro Treatment

Single cell suspensions of freshly extracted lymphoma cells were platedon an irradiated (20 Gy) feeder layer (300×10⁵ NIH-3T3 cells/10-cmplate) in 45% Iscove's modified Eagle medium, 45% Dulbecco's minimalessential medium, 10% fetal bovine serum, 100 U/ml penicillin andstreptomycin, 4 mM L-glutamine, and 25 μM 2-mercaptoethanol.

For western blot and immunofluorescence analysis, cells were treatedwith rapamycin 10 nM (Sigma) for 1 hour, then washed in PBS. Cytospinwas performed using 1×10⁵ cells at 500 rpm. Cells were then fixed in 4%paraformaldhyde and analyzed for phosphorylated S6 kinase using antibody9206 (Cell Signaling Technology, 1:200 dilution). For fluorescencedetection, an Axioscop 50 immunofluorescence microscope (Zeiss,Thornwood, N.Y.) was used.

Example 1 Generation of Eμ-myc Lymphoma with Defined Genetic Alterations

Lymphoma cells with defined genetic alterations were generated accordingto published procedures (see, e.g., Schmitt, C. A., et al., Cancer Cell,1(3):289-298, (2002)). Briefly, day 13.5-18.5 pregnant mice from anEμ-myc-transgenic to wildtype (C57BL/6) cross were sacrificed to obtainfetal livers, which were minced and grown at approximately 3×10⁶cells/ml in conditions supporting hematopoietic stem cell (HSC) growth(37% DMEM, 37% Iscove's modified Dulbecco's Medium [Gibco], supplementedwith 20% fetal calf serum, 2% L-glutamine [200 mM], 100 U/mlpenicillin/streptomycin, 5×10⁻⁵ M 2-mercaptoethanol, 4% 0.45 μm filteredWEHI-3B supernatant, 0.2 ng/ml recombinant murine interleukin-3, 2 ng/mlrecombinant murine interleukin-6, and 20 ng/ml recombinant murine stemcell factor [all cytokines from Research Diagnostics] at 37° C. in ahumidified 5% CO₂ atmosphere).

Eμ-myc/p53+/− HSCs were derived from crosses of Eμ-myc and p53+/− miceand detection of loss of heterozygosity (LOH) in the p53 locus was byallele specific PCR (Schmitt, C. A., et al., “Dissecting p53 tumorsuppressor functions in vivo,” Cancer Cell 1: 289-298 (2002) andSchmitt, C. A., et al., “INK4a/ARF mutations accelerate lymphomagenesisand promote chemoresistance by disabling p53,” Genes Dev. 13: 2670-2677(1999)).

Production of retroviral supernatants and transductions were carried outas previously described (Schmitt, C. A. and S. W. Lowe, Blood Cells MolDis 27(1):206-216, (2001)). High-titer retroviral supernatant was passedthrough a 0.45 μm filter and supplemented with 4 μg/ml polybrene(Sigma). About 6×10⁶ cells were infected four times by spinoculation at600 g for 10 min in 3 ml of retroviral supernatant every 6-8 hours.Twenty-four hours after the last infection, the fraction of GFPexpressing cells was measured by flow cytometry (FACScalibur, BectonDickinson), and typically was between 5% and 20%. Protein expression ofthe Akt construct was detected by western blot analysis using antibodiesagainst Phospho-Thr308 Akt (9275, Cell Signaling Technology, 1:1000dilution) and antibodies against HA (MMS-101P, Covance, 1:1000dilution). α-Tubulin (B-5-1-2, Sigma, 1:5000 dilution) served as aloading control. Cells were used for reconstitution of recipient mice 2days following the last infection.

For bone marrow reconstitution experiments 6- to 8-week old C57BL/6recipient mice received a single 10 Gy-dose of total-body irradiation(¹³⁷Cesium source; 0.8 Gy/min), and were reconstituted 6 hours laterwith approximately 3×10⁶ viable fetal liver cells by tail veininjection. Mice were housed on autoclaved bedding in air-filtered cagesand received neomycin-containing drinking water. Nonreconstituted,lethally irradiated mice were included as controls in every experiment,and typically had to be sacrificed between day 12 and 18 postirradiation due to bone marrow aplasia.

Upon the appearance of well-palpable lymphomas, tumors were harvestedand either fixed for histological evaluation, rendered single cellsuspensions and frozen in 10% DMSO or transplanted directly into normalmice for treatment studies (Schmitt, C. A., et al., “Genetic analysis ofchemoresistance in primary murine lymphomas,” Nat. Med. 6: 1029-1035(2000)).

The retroviral vectors used were MSCV-GFP (the empty vector control),MSCV-Bcl-2, MSCV-Akt (myrAkt), MSCV-Akt (K179A), and MSCV-eIF4E. For invivo competition experiments, MSCV-eIF4E was transduced into a smallpercentage of Akt lymphoma cells during a brief in vitro passage, andthe mixed population was re-injected into several recipient mice.

pMSCV-IRES-GFP was constructed by placing GFP downstream of the pCITE1internal ribosome entry site from encephalomycocarditis virus (IRES;Novagen) and cloning it into the EcoRI and SalI sites of the murine stemcell virus pMSCVneo (Clontech), replacing PGK and the neomycinresistance gene expressed in pMSCVneo (Van Parijs, L. et al., Immunity11:281-288, (1999)). A gene encoding an HA-tagged, myristoylated Akt (agift from B. A. Hemmings; Andjelkovic, M., et al., J. Biol. Chem.272(50):31515-31524, (1997); Andjelkovic, M. et al, Proc. Natl. Acad.Sci. USA 93(12):5699-5704, (1996)) and a gene encoding a kinase-deadmutant Akt (obtained by mutating residue K179 to A179) were subclonedinto pMSCV-IRES-GFP at the EcoRI and BglII sites. Thus,pMSCV-Akt-IRES-GFP includes the 5′ and 3′ long terminal repeats (LTRs)from pMSCV, the ψ⁺ extended packaging signal, the human Akt gene, IRES,GFP, an origin of replication from pUC, and a gene encodingbeta-lactamase, providing for ampicillin resistance. (See FIG. 4).

pMSCV-Bcl-2-IRES-GFP (MSCV-Bcl-2) has been described [Schmitt, C. A. andS. W. Lowe, Blood Cells Mol Dis 27(1):206-216, 2001]. To makepMSCV-Bcl-2-IRES-GFP, Bcl-2 was cloned into the EcoRI and BglII sites.

pMSCV-eIF4E-IRES-GFP was constructed as described above forpMSCV-IRES-GFP. A gene encoding eIF4E (GenBank Accession # XM_(—)371067)was subcloned from pGEX-6p1-meIF4E-wt (where m is murine), and cut withXhoI, klenowed the end, ligated EcoRI linkers (NEB) to the end,re-ligated and subsequently cut out the 680 bp (all coding region)fragment. This was ligated and subcloned into pMSCV-IRES-GFP at theEcoRI site of the MSC.

Other vectors, such as pMSCV-PTEN-IRES-GFP, could be similarlyconstructed using the above protocol to subclone PTEN into thepMSCV-IRES-GFP vector with the 5′ and 3′ LTPs from pMSCV by insertingflanking sites at either end of PTEN and ligating it into a site of MSCsuch as EcoRI or BglII. This method could also be used to subclone othergenes into similar vectors to induce altered gene expression in vivo, byinserting the appropriate flanking sites to cut out and re-ligate intothe pMSCV-IRES-GFP vector as needed.

The animals were monitored 2-3 times weekly for the occurrence ofwell-palpable peripheral lymph node enlargements. The time to onset datawere calculated from the time of reconstitution to lymphoma onset.Statistical evaluation of tumor onset data was by log-rank (Mantel-Cox)test for comparison of the Kaplan-Meier event-time format.

FIG. 2A shows the tumor onset in mice reconstituted with Eμ-myc HSCstransduced with control pMSCV (n=40), Akt (n=18), or Bcl-2 (n=18). Anaccelerated tumor onset is shown in both Akt-expressing andBcl-2-expressing mice. Morphologically, the lymphomas resemble theEμ-myc lymphomas, in that they are highly aggressive, predominantlylarge cell lymphomas, with a tendency for early dissemination,infiltration of non-lymphoid organs, and the frequent occurrence of anaccompanying leukemia. A kinase-dead mutant Akt did not acceleratelymphomagenesis, and Akt did not cause tumor formation in non-transgenicHSCs (data not shown).

Example 2 Tumor Free Survival of Akt-Expressing Mice in Response ofSecondary Tumors to Combination Therapy

Parent vector pMSCV-IRES-GFP, or derivative vectors pMSCV-Akt-IRES-GFPor pMSCV-Bcl-2-IRES-GFP were used to transduce hematopoietic stem cellsobtained as described above. Irradiated recipient mice were administeredthe transduced hematopoietic stem cells as described above. The schemeis presented in FIG. 1A.

Stem cells were infected using retroviral vectors (either pMSCV-IRES-GFPas control, pMSCV-Akt-IRES-GFP or pMSCV-Bcl-2-IRES-GFP). When primarytumors arose in these animals, the mice were sacrificed and the tumorswere harvested and injected into five recipient mice. Followinginjection of 10⁶ cells into the tail vein, upon development of palpable(secondary) tumors (ca. 3 weeks following the iv injection), mice weretreated with either adriamycin (10 mg/kg in H₂O, once, by i.p.injection) or rapamycin (4 mg/kg by i.p. injection) and adriamycin (d1rapamycin, d2 both drugs by separate injection, d3-5 rapamycin). Micewere then palpated twice weekly for disappearance or recurrence oftumors. Data are presented as Kaplan-Meier plots in FIGS. 5A-5D and showthe tumor free survival/time to relapse.

-   -   FIG. 5A: Pooled data from the first 8 mice in each treatment        group, receiving adriamycin treatment as described above. The        tumors are from more than 5 separate primary tumors.    -   FIG. 5B: Data from mice were stratified according to the        genotypes.    -   FIG. 5C: Treatment of Bcl-2 tumors with either adriamycin, or        adriamycin and rapamycin compared to the control tumors.    -   FIG. 5D: Treatment of Akt expressing tumors with adriamycin, or        combination of adriamycin and rapamycin.

Data presented in FIG. 6 are from the same mice treated as describedabove, but also include experiments using rapamycin as a single agent(same dose as above i.p. administration on days 1-5). Data are presentedas percentage of treated animals that achieved a complete remission.

Example 3 Variations in Response to Treatment on Identical Tumors

Primary tumors were produced as described above. Identical primarytumors were injected into 5 recipient animals, thus forming cohorts ofmice carrying identical tumors. The treatment regimens of 7 primary Akttumors (#135, #136 etc.) injected into multiple recipient mice wereanalyzed as a matched group in which identical secondary tumors receiveddifferent treatments. The treatments were: adriamycin, rapamycin and thecombination rapa-adr (schedule as above in Example 2); the treatmentshown in FIG. 7 as adr-rapa is adriamycin and rapamycin given on day 1,days 2-5 rapamycin alone at the previous doses by i.p. injection. Micewere monitored twice weekly by palpation and blood smears. The graphindicates the individual tumor free survival times, as time to relapsemeasured in days. If the mice were never tumor free, time to relapse isscored as ‘0’ days.

Example 4 Green Fluorescent Protein Imaging of Tumors in Mice

Paired Eμ-myc/Akt secondary tumors were produced by injection of anAkt-GFP expressing primary tumor into 3 recipient animals. The animalswere treated as described with either adriamyin d1, rapamycin d1-5 orrapa d1, adr+rapa d2 and rapa d3-5. The green fluorescent proteinimaging technique has been described elsewhere (M. Yang et al., ProcNatl Acad Sci USA 97, 1206-11 (2000); M. Yang et al., Clin Cancer Res 5,3549-59 (1999); M. Yang et al., Cancer Res 59, 781-6 (1999); M. Yang etal., Cancer Res 58, 4217-21 (1998); M. Yang, E. Baranov, A. R. Moossa,S. Pemnan, R. M. Hoffman, Proc Natl Acad Sci USA 97, 12278-82 (2000);and C. A. Schmitt et al., Cancer Cell 1, 289-98 (2002)). Images taken onday 21 post treatment revealed sizable tumors at several sites for bothanimals treated with either adriamycin or rapamycin (FIG. 9B). Wholebody imaging of these animals treated with either adriamycin orrapamycin alone revealed that splenic infiltration, thoracic walllymphoma, cervical lymphoma and inguinal lymphoma persisted after 21days following treatment. In marked contrast, the combination ofchemotherapy and rapamycin showed potent activity against Akt-expressinglymphomas. The animal treated with the combination of drugs appeared tobe free of tumors.

Example 5 TUNEL and Ki67 Staining of Histological Sections from Akt andBcl-2 Tumors

Untreated tumors produced using pMSCV-IRES-GFP (Eμ-myc/wt),pMSCV-Akt-IRES-GFP (Eμ-myc/Akt) or pMSCV-Bcl-2-IRES-GFP (Eμ-myc/Bcl-2)were fixed in neutral buffered formaldehyde (4% in PBS) for 24 hrs, thentransferred into neutral PBS. Sectioning was by standard microtome.Immunohistochemical (IHC) staining was done by addition of a) TUNELreagent to demonstrate apoptotic cells and b) Ki67 as a marker ofproliferation, using NCL-Ki67p antibody from Novocastra (Vector Labs) at1:5000.

As shown in FIG. 2C, cells of all three genotypes showed extensivestaining with antibodies to the Ki67 antigen, indicating a high rate ofproliferation. In contrast, TUNEL analysis showed much less DNAfragmentation or apoptosis occurring in Eμ-myc/Akt or Eμ-myc/Bcl-2 cellsrelative to Eμ-myc/wt control cells.

Example 6 36-Kinase Inhibition Following Rapamycin Treatment In Vivo

Cells for this immunofluorescent antibody binding study were derivedfrom the offspring of a cross of an Eμ-myc mouse to a PTEN^(+/−) mouse.These mice develop Eμ-myc/PTEN^(+/−) tumors. Cells of these tumors canbe grown in culture, unlike Eμ-myc/Akt cells. Eμ-myc/PTEN^(+/−) tumorcells were incubated with 10 nM rapamycin (Sigma) for 1 hour, thenwashed in PBS. Cytospin was performed using 1×10⁵ cells at 500 rpm.Cells were then fixed in 4% paraformaldhyde for 15 minutes, washed andincubated with primary antibody to S6-kinase (Cell SignalingTechnology), then washed and incubated with secondary antibody. Forfluorescence detection, we used an Axioscop 50 immunofluorescencemicroscope (Zeiss, Thornwood, N.Y.). DAPI (4′,6-diamidino-2-phenylindoledihydrochloride; Sigma) was added with the antibody to stain all cells(blue stain). Untreated cells fluoresced brightly, but cells treatedwith rapamycin showed almost no fluorescence. FIG. 24 shows the markedinhibition of S6-kinase (red staining) following rapamycin treatment invitro.

Insert Immunofluorescent Figure of PTEN Cells Treated with RAP

Example 7 Western Blots were Used to Test for the Effect of Rapamycin onPhosphorylation of Elongation Factor eIF4G, a Target of mTOR

Cells of one primary lymphoma were used to inject three recipient mice,and secondary lymphomas were allowed to develop. The mice were treatedwith 1) nothing, as control, 2) rapamycin at 4 mg/kg, or 3) rapamycin at4 mg/kg and cyclophosphamide at 300 mg/kg i.p.).

A control experiment was performed using 3T3 fibroblasts. Fibroblastswere incubated in 1) medium without serum (cells serum-starved for 48hours), 2) medium with serum (known to activate the PI3 kinase/Aktpathway) or 3) medium with serum, with addition of 10 nM rapamycin for 1hour.

Protein extracts were prepared, samples were separated byelectrophoresis on SDS-polyacrylamide gels, and the proteins weretransferred to a membrane for probing with antibodies againstphospho-eIF4G (Cell Signaling Technology).

Both the rapamycin alone and the rapamycin plus cyclophosphamidecombination treatment inhibited phosphorylation of elongation factoreIF4G in the lymphoma cells. Rapamycin also inhibited phosphorylation ofelongation factor eIF4G in 3T3 fibroblasts. Growth in serum increasedthe level of phosphorylated eIF4G compared to the level ofphosphorylated eIF4G in 3T3 cells grown without serum (see FIG. 8A).

Example 8 Akt and Bcl-2 Accelerate Lymphomagenesis and Cause DrugResistance In Vivo

See “In Vivo Chemotherapy” above for procedures. Mice reconstituted withEμ-myc transgenic HSCs transduced with pMSCV (“empty vector”), Akt orBcl-2 were followed for time to development of lymphomas. Like Bcl-2(Schmitt, C. A., et al., “Genetic analysis of chemoresistance in primarymurine lymphomas,” Nat. Med. 6: 1029-1035 (2000)), Akt dramaticallyaccelerated lymphomagenesis relative to controls (FIG. 2, p<0.0001 forboth Akt and Bcl-2 relative to MSCV). Of note, a kinase-dead mutant Akt(K179A) did not cause acceleration of lymphogenesis, and Akt did notcause tumor formation in non-transgenic HSCs.

Sections of Eμ-myc/Akt lymphomas were stained with hematoxylin and eosin(H/E), and immunohistochemical (IHC) stains for B220/CD45R andphosporylated Akt. Compared to lymphomas arising in Eμ-myc mice, the Aktand Bcl-2 lymphomas were more invasive and often associated withleukemia (FIG. 2D, data not shown). Microphotographs of H/E stainedliver sections revealed perivascular infiltration in control tumors andparenchymal invasion in Akt and Bcl-2 tumors. Immunophenotyping revealedthat Akt and Bcl-2 lymphomas were derived from a B220/CD45R-positiveprogenitor cell which lacked other markers of B-cell differentiation,whereas control lymphomas were typically derived from a more matureB-cell type (see FIG. 2B). As shown in FIG. 15, representative flowcytometric immunophenotyping of control (myc), Bcl-2 (myc/Bcl-2), Akt(myc/Akt) and eIF4E (myc/eIF4E) tumors, tumor cells were gated on thebasis of forward scatter and side scatter and GFP positivity. Both Aktand Bcl-2 tumors did not express B-cell antigens other than CD45R (B220)but were positive for CD4, whereas the control and eIF4E tumors had amature B-cell marker profile. At least three tumors of each genotypewere analyzed. See Table 2. TABLE 2 Immunophenotype of tumors expressingEμ-myc (Control), Eμ-myc/Bcl-2, Eμ-myc/Akt and Eμ-myc/eIF4E AntigenControl Bcl-2 Akt eIF4E B-cell antigens CD45R Pos Pos Pos Pos CD19 PosNeg Neg/Weak Pos IgM Pos Neg Neg Variable IgK Pos Neg Neg Pos CD23(FcεR)Neg Neg Neg Neg CD138 Variable Neg/Weak Neg Variable T-cell antigens NegCD3 Neg Neg Neg Neg TCR α/β Neg Neg Neg Neg TCR γ/δ Neg Neg Neg Neg CD4Neg Pos Pos Neg CD5 Neg Neg Pos Neg CD8a Neg Neg/Weak Neg VariableMyeloid antigens Ly-6G (Gr-1) Neg Pos Neg Neg CD11b (Mac-1) Neg/WeakNeg/Weak Weak Variable Ly-76 (TER119) Neg Neg Neg Neg Ly-71 (F4/80) NegNeg Pos Neg CD41 Neg Neg Neg Neg Lineage non-specific antigens CD45 PosPos Pos Pos Early antigens CD34 Neg Neg Neg/Weak Neg Ly-6A/E (Sca-1) NegPos Pos Neg CD90 (Thy1) Variable Pos Variable Neg CD117 (c-Kit) Neg NegNeg Neg Additional antigens CD16/32 (FCγRII/III) Pos Variable Pos PosCD31 (PECAM) Pos Pos Pos Pos CD43 Pos Pos Variable Pos CD59 (Ly-6C)Neg/Weak Pos Pos Variable CD69 Neg Neg Neg Pos CD71 (Transferrin Pos PosPos Pos Receptor) CD86 Variable Pos Pos Pos Class II (I-a) Pos Pos PosPos

Although lymphomas of all genotypes proliferated at similar rapid ratesas assessed by Ki-67 staining, Akt and Bcl-2 lymphomas showed much lessapoptosis relative to controls as measured by TUNEL (see FIG. 2C). Theimpact of Akt on tumor behavior was reminiscent of p53 tumor suppressorgene loss, which normally acts to limit Myc-induced lymphomagenesis bypromoting apoptosis (Schmitt, C. A., et al., “Genetic analysis ofchemoresistance in primary murine lymphomas,” Nat. Med. 6: 1029-1035(2000)). In fact, whereas all lymphomas arising from p53+/− Eμ-mychematopoietic stem cells lost the wild-type p53 allele, those expressingeither Bcl-2 or Akt did not (see FIG. 16). Tumorigenesis and acts in amanner that is temporally and pathologically similar to a strictlyanti-apoptotic gene.

C57BL/6 mice harboring different transplanted lymphomas were treatedwith cyclophosphamide (Cytoxan, CTX) or doxorubicin (DXR) and monitoredfor tumor free and overall survival (see FIGS. 2E and F and FIG. 17).Here, animals harboring ARF-null lymphomas were used as controls, sincethese lymphomas are also highly aggressive but remain chemosensitive(Schmitt, C. A., et al., “Dissecting p53 tumor suppressor functions invivo,” Cancer Cell 1: 289-298 (2002)). Following CTX therapy, miceharboring control lymphomas invariably entered a complete remissionand >50% remained tumor free after 100 days. In contrast, mice harboringAkt lymphomas responded poorly, typically displaying remissions of lessthan 20 days with no long-term survivors (p<0.001 relative to controllymphomas). Although DXR produced fewer long lasting remissions in thecontrol cohort than CTX, mice harboring Akt lymphomas still displayedsubstantial reductions in tumor free and overall survival (FIG. 2F andFIG. 17B, p<0.0001 for Akt and Bcl-2 vs. control). Therefore,Akt-mediated survival signaling can promote drug resistance in vivo.

Example 9 Inhibition of mTOR Sensitizes Akt Tumors to DNA Damage-InducedApoptosis

We asked whether pharmacologically inhibiting the pathway downstream ofAkt might have anti-tumor effects. One established Akt effector is mTOR,a serine/threonine kinase implicated in translation control, which canbe potently inhibited by the immunosuppressant rapamycin (reviewed inHuang, S. and Houghton, P. J., “Targeting mTOR signaling for cancertherapy. Curr Opin. Pharmacol. 3: 371-377 (2003)). Although notconsidered a primary component of Akt survival signaling, mTOR canmediate metabolic changes important for cell survival in growth factorpoor environments (Plas, D. R., et al., “Akt and Bcl-xL promote growthfactor-independent survival through distinct effects on mitochondrialphysiology,” J. Biol. Chem. 276: 12041-12048 (2001) and Edinger, A. L.and Thompson, C. B., “Akt maintains cell size and survival by increasingmTOR-dependent nutrient uptake,” Mol. Biol. Cell 13: 2276-2288 (2002)).Rapamycin has modest anti-tumor activity against PTEN-deficient tumorsin animal studies and can potentiate drug-induced cell death in vitro(Neshat, M. S., et al., “Enhanced sensitivity of PTEN-deficient tumorsto inhibition of FRAP/mTOR,” Proc. Natl. Acad. Sci. USA 98: 10314-10319(2001); Grunwald, V., et al., “Inhibitors of mTOR reverse doxorubicinresistance conferred by PTEN status in prostate cancer cells.” CancerRes. 62: 6141-6145 (2002); Podsypanina, K., et al., “An inhibitor ofmTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/−mice,” Proc. Natl. Acad. Sci. USA 98: 10320-10325 (2001); and Hosoi, H.,et al., “Rapamycin causes poorly reversible inhibition of mTOR andinduces p53-independent apoptosis in human rhabdomyosarcoma cells,”Cancer Res. 59: 886-894 (1999)). We therefore asked whether rapamycinwas effective against our defined murine lymphomas, and whether itsactivity was genotype-dependent or enhanced by chemotherapy.

To determine whether rapamycin inhibits mTOR in Eμ-myc lymphomas, itsactivity was indirectly assessed in extracts from untreated or treatedlymphomas using antibodies that specifically recognize thephosphorylated forms of two mTOR targets, the ribosomal S6 protein(which is phosphorylated by the S6 kinase) and eIF4G.

Animals harboring lymphomas of the indicated genotypes were either nottreated or treated with DXR, rapamycin or DXR and rapamycin. Lymphomacells were harvested 7 hours post treatment. Lysates were subjected toimmunoblotting for phosphorylated and total ribosomal S6 protein,phosphorylated and total eIF4G, Akt, Bcl-2, and α-tubulin as a loadingcontrol. The treated and untreated lymphomas were TUNEL-stained forapoptotic cells. As expected, untreated Akt lymphomas expressedsubstantially more phosphorylated S6 and eIF4G relative to Bcl-2lymphomas (see FIG. 8A, compare lanes 1 and 5). Treatment with rapamycinalone or in combination with chemotherapy substantially reduced S6 andeIF4G phosphorylation in Akt lymphomas (compare lanes 1 to 3 and 4).Hence, rapamycin inhibits mTOR activity in vivo in a manner that is notaffected by conventional therapies.

Example 10 Rapamycin Reverses Apoptotic Defects and Chemoresistance inAkt Lymphomas

We examined the acute and long-term effects of rapamycin in vivo. In Aktlymphomas, rapamycin treatment produced only a slight increase inapoptosis relative to untreated counterparts by 6 hours post-therapy, asassessed by PARP cleavage in lymphoma extracts (see FIG. 8A, comparelanes 1 to 3) and by TUNEL staining of lymphoma sections (see FIG. 8B).Accordingly, these mice rarely achieved complete remissions (FIGS. 9Aand B) and showed tumor free and overall survival patterns that were nobetter than those produced by conventional therapy FIG. 9C and FIG. 11).Furthermore, rapamycin displayed even less activity against lymphomas ofother genotypes (FIGS. 8 and 9). Therefore, despite its ability toinhibit mTOR in vivo, rapamycin had only modest activity as a singleagent.

In marked contrast, the combination of chemotherapy and rapamycin showedpotent activity against Akt-expressing lymphomas. For example, DXR andrapamycin treatment produced PARP cleavage and massive apoptosis 6 hourspost-therapy (FIG. 8A, compare lanes 1 and 4; FIG. 8B), with all of theanimals achieving complete remissions (FIGS. 9A and B, see FIG. 10 for acomparison of matched groups). Indeed, DXR and rapamycin produced morecomplete remissions and better tumor free survival in the otherwise drugresistant Akt lymphomas than DXR produced in the chemosensitive controls(FIGS. 9A and C, p<0.001 for RAP+DXR vs. DXR or RAP alone), resulting ina substantial survival benefit (FIG. 11A, p<0.001 for RAP+DXR vs. DXR orRAP alone). Similar effects were observed with the combination of CTXand rapamycin, where approximately half of the mice achieved remissionslasting more than 60 days compared to none for either agent alone (FIGS.11B and 13A, p<0.001). Consequently, rapamycin reverses the apoptoticdefects and drug resistance occurring in Akt lymphomas.

Mice bearing matched tumors were generated by injecting 1×10⁶ tumorcells from the same primary tumor into 3 recipient animals. Eight‘matched groups’ of 3 mice received DXR, RAP, or DXR+RAP. Mice weremonitored twice weekly by palpation and blood smears. FIG. 10 indicatesthe individual tumor free survival times and the arithmetic mean; apaired t-test analysis comparing either combination to the single agentsconfirmed statistical significance (for DXR versus RAP+DXR: p=0.0002;RAP versus RAP+DXR: p<0.0001; while DXR versus RAP: p=0.7).

The potent effects of rapamycin in combination therapy were not observedin lymphomas of other genotypes. Bcl-2 lymphomas, which showed a similardrug resistance profile to either rapamycin or chemotherapy alone,remained non-responsive to the combination as assessed in both acute andlong-term assays (FIGS. 9A and D; FIGS. 11C and D; FIG. 12B). Moreover,control lymphomas, which were initially chemosensitive, showed noimprovement in short or long term responses following combinationtherapy and, in fact, rapamycin appeared to antagonize drug-inducedapoptosis in these cells (FIG. 8B). Therefore, while each lymphomastudied contained an anti-apoptotic lesion, the chemosensitizing effectsof rapamycin were specific for tumors with constitutive Akt signaling.

Example 11 eIF4E is Oncogenic In Vivo and Causes Resistance toConventional and Targeted Therapy

The results described above suggest that mTOR is essential forAkt-mediated survival signaling. mTOR can regulate translation inresponse to nutrients and growth factors by phosphorylating keycomponents of the protein synthesis machinery, including the 40Sribosomal protein S6 kinase, p₇₀ ^(S6K), and the 4E-BP proteins(Schmelzle, T. and Hall, M. N., “TOR, a central controller of cellgrowth,” Cell 103: 253-262 (2000)). Phosphorylation of 4E-BP, in turn,releases the translation initiation factor eIF4E to stimulatecap-dependent translation. Although its role in oncogenesis is poorlyunderstood, eIF4E can have transforming and anti-apoptotic activities invitro (Lazaris-Karatzas, A., et al., “Malignant transformation by aeukaryotic initiation factor subunit that binds to mRNA 5′ cap,” Nature345: 544-547 (1990) and Polunovsky, V. A., et al. “Translational controlof the antiapoptotic function of Ras,” J. Biol. Chem. 275: 24776-24780(2000)) and is overexpressed in many tumor types (Hershey, J. W. B. andMiyamoto, S. In: Translational Control of Gene Expression, N. Sonenberget al., eds., Cold Spring Harbor, N.Y., (2000) pp. 637-654).Furthermore, Akt-expressing tumors selectively increase the translationof some mRNAs in a rapamycin-reversible manner (Grolleau, A., et al.,“Global and specific translational control by rapamycin in T cellsuncovered by microarrays and proteomics,” J. Biol. Chem. 277:22175-22184 (2002) and Rajasekhar, V. K., et al., “Oncogenic Ras and Aktsignaling contribute to glioblastoma formation by differentialrecruitment of existing mRNAs to polysomes,” Mol. Cell. 12: 889-901(2003)). Therefore, to further examine the effects of translationalcontrol on tumor phenotypes, we directly compared eIF4E to Akt in theEμ-myc model (see FIG. 1).

The time to tumor development was observed, following hematopoieticreconstitution with Eμ-myc transgenic HSCs expressing eIF4E (n=15)versus control Eμ-myc transgenic HSCs infected with “empty vector” pMSCVand Eμ-myc transgenic HSCs transduced with Akt. (See FIG. 13A, samecontrol and Akt data from FIG. 2). eIF4E accelerated lymphomagenesis ina manner that was similar to Akt (FIG. 13A, median onset of 50 and 42.5days for Akt and eIF4E, respectively, p<0.0001 for eIF4E vs. control).Of note, eIF4E did not induce tumors in a non-transgenic backgroundwithin the observation period (>125 days, data not shown).

Organs (lymph node, liver, kidney/renal pelvis) from mice bearingEμ-myc/eIF4E lymphomas were sectioned and stained with hematoxylin andeosin (H/E) (FIG. 13B, top). Untreated tumors were examined by IHCstains for phosphorylated Akt and Ki-67, and by TUNEL, using sections oftumors harvested 6-8 hours following the therapy (FIG. 13B, bottom).eIF4E could also compensate for p53 loss during lymphomagenesis, aslymphomas arising from p53+/− Eμ-myc hematopoietic stem cells retainedthe wild-type p53 allele (FIG. 16). Despite these similarities, eIF4Elymphomas were derived from a more mature B-cell type and often producedan aggressive infiltration of the renal pelvis distinct fromAkt-associated pathologies (FIGS. 15 and 13B). Nevertheless, eIF4E isclearly a potent oncogene in vivo, producing phenotypes consistent withan anti-apoptotic gene.

Moreover, like Akt lymphomas, eIF4E lymphomas displayed a disseminatedpathology and a high proliferation/apoptosis ratio, despite only a ˜2.5fold increase in eIF4E protein (see FIG. 13C, compare lanes 1 to 3 andFIG. 14). Lysates of eIF4E and Akt tumors, harvested without priortherapy (U) or 6 hours after rapamycin (R) administration in vivo weresubjected to immunoblotting for eIF4E binding protein 1 (4E-BP1), thephosphorylated and total ribosomal S6 protein (p-S6 and S6), totaleIF4E, phosphorylated and total Akt (p-Akt and Akt) proteins, and actinas a loading control (see FIG. 13C). Note that untreated Akt tumors(lane 3) show an increase in the phosphorylated, slower migrating formsof 4E-BP1.

Allele-specific PCR was carried out to detect the wild-type p53 WT) andmutant allele (p53 Neo) in tumors derived from Eμ-myc/p53^(+/−) HSCs.All control tumors showed loss of heterozygosity (LOH) (8/8), while noneof the Akt (0/5), Bcl-2 (0/5) or eIF4E (0/3) tumors underwent LOH (seeFIG. 16).

In flow cytometric immunophenotyping of control (myc), Bcl-2(myc/Bcl-2), Akt (myc/Akt) and eIF4E (myc/eIF4E) tumors, tumor cellswere gated on the basis of forward scatter and side scatter and GFPpositivity. At least three tumors of each genotype were analyzed. BothAkt and Bcl-2 tumors did not express B-cell antigens other than CD45R(B220) but were positive for CD4, whereas the control and eIF4E tumorshad a mature B-cell marker profile (see FIG. 15).

Lysates derived from Bcl-2 (n=3), Akt (n=5) and eIF4E (n=5) tumors wereprobed with antibodies against eIF4E and β-actin as loading control and1125 labeled Protein A to quantitate eIF4E expression. Radioactivity wasquantitated on western blots by phosphorimaging. FIG. 14 is a graphicrepresentation of eIF4E expression relative to Akt and Bcl-2 tumors. Thepresented data are the combined averages of 4 independent probings ofthe tumor set with error bars denoting the standard deviation. Bcl-2(n=4), Akt (n=5), eIF4E (n=5).

Lymphomas expressing eIF4E were highly resistant to DXR therapy relativeto controls, and mice harboring these lymphomas displayed tumor free andoverall survival patterns that were indistinguishable from thoseharboring Akt lymphomas (FIG. 13E, p<0.0001 vs. Control; p=0.47 vs.Akt). However, whereas Akt lymphomas displayed high mTOR activity asassessed by an increase in the rapamycin-sensitive phosphorylation of S6and 4E-BP1, eIF4E lymphomas did not (FIG. 13C). However, in contrast toAkt lymphomas, eIF4E lymphomas displayed no increase in therapamycin-sensitive S6 phosphorylation and 4E-BP1 (FIG. 13C) and werenon-responsive to rapamycin/DXR therapy in vivo (FIG. 13E, FIG. 11E,p<0.0001). Furthermore, introduction of eIF4E into an initiallysensitive Akt lymphoma conferred resistance to rapamycin andchemotherapy; hence cells co-expressing eIF4E were enriched in mixedpopulations following therapy relative to over those expressing Aktalone (FIGS. 13F and G). Together, these results indicate that eIF4E canrecapitulate Akt action in oncogenesis and drug resistance. They alsoshow that eIF4E can confer resistance to a rapamycin based therapy invivo, presumably because it acts downstream of mTOR.

Example 12 Rapamycin Reverses Apoptotic Defects and Chemoresistance inPTEN-Deficient Lymphomas

Generation and development of PTEN mutants (see Di Cristofano A., etal., “Pten is essential for embryonic development and tumorsuppression”, Nature Genetics, 19(4):348-355 (1998); Suzuki A., et al.,“High cancer susceptibility and embryonic lethality associated withmutation of the PTEN tumor suppressor gene in mice”, Current Biology,8(21):1169-1178, (1998)).

Eμ-myc/PTEN+/− mice were derived from a cross of a transgenic Eμ-mycmouse to a transgenic PTEN+/− mouse and detection of loss ofheterozygosity (LOH) in the PTEN locus was by allele specific PCR (DiCristofano A., et al., Nature Genetics, 19(4):348-355, 1998).

The time from birth to tumor development was observed in Eμ-myc/PTEN+/−mice (n=12) and control Eμ-myc/PTEN+/+ mice (n=24). The animals weremonitored 2-3 times weekly for the occurrence of well-palpableperipheral lymph node enlargements. Tumors arising in Eμ-myc/PTEN+/−mice were harvested and either fixed for histological evaluation,rendered single cell suspensions and frozen in 10% DMSO or transplanteddirectly into normal C57BL/6 (non-transgenic) mice for treatment studies(Schmitt, C. A., et al., “Genetic analysis of chemoresistance in primarymurine lymphomas,” Nat. Med. 6: 1029-1035 (2000)).

Initial time to tumor onset in Eμ-myc/PTEN+1- and Eμ-myc/PTEN+/− mice isshown in FIG. 18. Time to onset data were calculated from birth tolymphoma onset (cumulative survival versus time). Statistical evaluationof tumor onset data was by log-rank (Mantel-Cox) test for comparison ofthe Kaplan-Meier event-time format. Results show that PTEN heterozygousmice are highly susceptible to tumors, as heterozygosity caused asignificant acceleration and increased penetrance in lymphomadevelopment in vivo (p<0.005).

Primary tumors arising in Eμ-myc/PTEN+/− and Eμ-myc/PTEN+/− mice wereharvested and injected into C57BL/6 (non-transgenic) mice. Upon tumorformation of palpable secondary tumors, mice were treated with a singlei.p. administration of drug. The treatment schedule included aninjection with either DXR (10 mg/ml in 0.9% saline), CTX (300 mg/kg in0.9% saline), RAP (4 mg/kg in 0.9% H₂O with 5% PEG/TWEEN), or acombination thereof (d1: DXR or CTX, d2: DXR or CTX and RAP, d3-5: DXRpr CTX). Mice were then palpated twice weekly for disappearance orrecurrence of tumors. Data are presented as Kaplan-Meier plots in FIG.19, and show the tumor free survival/time to relapse. Time to relapsefor mice that were never tumor free was scored as ‘0’ days. Tumor freesurvival implies the complete absence of detectable disease, i.e. nofrank leukemia and no palpable tumors, a relapse denotes the recurrenceof either, e.g. following RAP+DXR treatment (green line), all miceachieve a complete remission, by day 15 the first animal has relapsedand by day 23 all mice have relapsed. The bottom panel has an equivalentanalysis for treatment with CTX, RAP, and RAP+CTX combination. Togetherthese data show that Eμ-myc/PTEN+/− tumors are resistant to DXR and RAPreverses this drug resistance.

Example 13 eIF4E Blocks Rapamycin's Effect on Chemoresistance inPTEN-Deficient Lymphomas

Parent vector pMSCV-IRES-GFP, or derivative vector pMSCV-eIF4E-IRES-GFPwere used to transduce hematopoietic stem cells obtained as described inprior examples. Recipient Eμ-myc/PTEN+/− and Eμ-myc/PTEN−/− mice wereadministered the transduced hematopoietic stem cells as describedpreviously.

Stem cells were infected using retroviral vectors (either pMSCV-IRES-GFPas control, or pMSCV-eIF4E-IRES-GFP). When primary tumors arose in theseanimals, the mice were sacrificed and the tumors were harvested andinjected into recipient C57BL/6 (non-transgenic) mice. Followinginjection of 10⁶ cells into the tail vein, upon development of palpable(secondary) tumors (approximately 3 weeks following the i.v. injection),drugs were administered. Mice were treated once by i.p. injection witheither DXR (10 mg/ml in 0.9% saline), CTX (300 mg/kg in 0.9% saline),RAP (4 mg/kg in 0.9% H₂O with 5% PEG/TWEEN), or a combination whereinthe combination schedule was d1: DXR or CTX, d2: DXR or CTX and RAP,d3-5: DXR pr CTX). The tumors were harvested 48 hours after treatmentand a single cell suspension was analyzed for GFP expression by flowcytometry as described previously. GFP expression denotes tumor cellswhich are productively infected with MSCV-eIF4E-IRES-GFP.

FIG. 20 shows the GFP content measured by flow cytometry under eachtreatment condition (untreated, DXR, RAP, RAP+DXR). The observedincrease in GFP-positive cells in all treated tumors as compared to theuntreated control indicates an advantage/resistance of eIF4E-infectedcells under different therapies.

Example 14 Tumor Free Survival of Different Forms of Mutated eIF4E CellsInfluence eIF4E's Oncogenicity In Vitro

Day 13.5-18.5 pregnant mice from an Eμ-myc-transgenic to wildtype(C57BL/6) cross were sacrificed to obtain fetal livers, which wereminced and grown at approximately 3×10⁶ cells/ml in conditionssupporting hematopoietic stem cell (HSC) growth (37% DMEM, 37% Iscove'smodified Dulbecco's Medium [Gibco], supplemented with 20% fetal calfserum, 2% L-glutamine [200 mM], 100 U/ml penicillin/streptomycin, 5×10⁻⁵M 2-mercaptoethanol, 4% 0.45 μm filtered WEHI-3B supernatant, 0.2 ng/mlrecombinant murine interleukin-3, 2 ng/ml recombinant murineinterleukin-6, and 20 ng/ml recombinant murine stem cell factor [allcytokines from Research Diagnostics] at 37° C. in a humidified 5% CO₂atmosphere).

Eμ-myc HSCs were harvested and infected in vitro, as described earlier,with MSCV-IRES-GFP constructs expressing different mutants of eIF4E. Themutants were: (a) E103A; an amino acid involved in ionic interactionwith the guanosine ring (bearing a partial positive charge near the N-7methyl portion of the guanosine ring), (b) S209A; which alters a MNKphosphorylation site (see Pyronnet S. et al., “Human eukaryotictranslation initiation factor 4G (eIF4G) recruits Mnk1 to phosphorylateeIF4E”, EMBO J. 18, 270-279 (1999)). Such a mutation does not affect theformation of initiation complex, mitogen-stimulated increase incap-dependent translation, and cell proliferation. It is thought thatthrough the kinase activity of MNKs, eIF4E activity is limited underphysiological conditions. MNK1 may define a convergence point betweenthe growth factor-activated and one of the stress-activated proteinkinase cascades and is a candidate to phosphorylate eIF4E in cells, (c)W56A; which should not bind the cap-structure, (d) W73A; an importantresidue in the interaction of eIF4E and eIF4E binding protein (4E-BP).Changing this amino acid is known to prevent productive interaction with4E-BP and eIF4G.

FIG. 21 shows that E103A induces tumors with high penetrance and onlyslightly slower than wild type eIF4E, while the mutants W56A and W73Aappear to be inactive. Moreover, S209A induces tumors at lowerpenetrance and much delayed. This would indicate that the phosporylationsite S209 is important for eIF4E's oncogenicity. As phosphorylation atthis site is mediated via p38 and MEKK activation of MNK-1, this findingalso indicates that eIF4E tumors (and similarly Akt expressing tumors)should be sensitive to pharmacological inhibitors of these kinases.

Example 15 Presence of Novel PTEN Short Hairpins Induces Chemoresistanceand Downregulation of PTEN Expression In Vitro

Retroviral-mediated gene transfer was performed using Phoenix packagingcells (a gift from G. Nolan, Stanford University) as previouslydescribed (Serrano M., et al., “Oncogenic ras provokes premature cellsenescence associated with accumulation of p53 and p161NK4A”, Cell88:593-602, (1997)). The two murine PML hairpins (SEQ ID NO:1 and SEQ IDNO:2) were cloned into MSCVpuro (Clontech) as previously described(Hemann M. T., et al., “An epi-allelic series of p53 hypomorphs createdby stable RNAi produces distinct tumor phenotypes in vivo”, NatureGenetics 33:396-400, (2003)). Infected cell populations were selected inpuromycin (2 μg/ml, 2 days).

Initial time to tumor onset in Eμ-myc mice infected with a short-hairpinagainst PTEN (MSCV-shPTEN-IRES-GFP) is shown in FIG. 22. Time to onsetdata were calculated following reconstitution of lethally irradiatedmice with HSCs from the fetal livers of Eμ-myc mice infected with ashort hairpin against two mPTEN sequences (PTEN sh1 and PTEN sh2) (n=5).Data is presented as cumulative survival versus time. Control group areequivalent HSCs infected with a vector encoding only GFP (MSCV-IRES-GFP)(n=25). Statistical evaluation of tumor onset data was by log-rank(Mantel-Cox) test for comparison of the Kaplan-Meier event-time format.Results show that shPTEN induces lymphomas in the Eμ-myc mouse model.

Wild type Mouse Embryo Fibroblasts (wt MEFs) containing a control vector(MSCV-IRES-GFP) or a short hairpin against PTEN sh1 and PTEN sh2 werecollected and analyzed for the expression of endogenous total andphosphorylated PTEN by western blotting. Immunoblots were performed fromwhole-cell lysates obtained by boiling cell pellets solubilized inLaemmli sample buffer (de Stanchina E., et al., “E1A signaling to p53involves the p19ARF tumor suppressor.”, Genes Dev., 12:2434-2442,(1998)). Samples of 30 mg of protein (Bio-Rad protein assay) wereseparated on SDS-PAGE gels and transferred to Immobilon-P membranes(Millipore). The antibodies used were: anti-PTEN 488 (1:1000 dilution, agift from M. Myers) and anti-PTEN phosphoSer380 (1:1000 dilution, CellSignaling). Proteins were visualized using ECL (Amersham) or SuperSignalWest Femtomaximum (Pierce). As shown in FIG. 23, PTEN expression isdownregulated in the presence of PTEN short hairpins.

Example 16 Tumor Free Survival of eIF3E-Expressing Mice In Vivo

Retroviral infections using MSCV vectors were carried out as describedpreviously. Infected HSC populations were propagated in recipient miceas described previously. Parent vector pMSCV-IRES-GFP was constructed asdescribed previously. Derivative vector pMSCV-eIF3E-IRES-GFP wasconstructed with a gene encoding the p48 fragment of eIF3E. The eIF3Egene (GenBank accession #: 7: XM_(—)129062) was cloned from pMV7 byexcising EcoRI and HindIII sites of pMV7 (Mayeur, G. L. and Hershey, W.B., “Malignant transformation by the eukaryotic translation initiationfactor 3 subunit p48 (eIF3E)”, FEBS Letters, 514: 49-54 (2002)).MSCV-IRES-GFP was then cut with these same enzymes (and as describedpreviously) and eIF3E was ligated into MSCV.

The tumor free survival of Eμ-myc mice infected with p48/eIF3E(MSCV-p48/eIF3E-IRES-GFP) is shown in FIG. 25. Time to onset data werecalculated following reconstitution of lethally irradiated mice withHSCs from the fetal livers of Eμ-myc mice infected with a short hairpinagainst p48/eIF3E (n=16). Data is presented as cumulative survivalversus time. Control group are equivalent HSCs infected with a vectorencoding only GFP (MSCV-IRES-GFP) (n=25). Statistical evaluation oftumor onset data was by log-rank (Mantel-Cox) test for comparison of theKaplan-Meier event-time format. Results show that p48/eIF3E induceslymphomas in the Eμ-myc mouse model.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended embodiments. Mouse PTEN hairpin 1AAAAAAGTCAAGTCTAAGCCGAATCCACCCCCTCGCAAG SEQ ID NO:1CTTCCAAGAGGATGGATTCGACTTAGACTTGACGGTGTT TCGTCCTTTCCACAA Mouse PTENhairpin 2 AAAAAAGGGCCCTGAATTAGAAGAATACATCTTCACAAG SEQ ID NO:2CTTCTGAAGATATATTCCTCCAATTCAGGAC

1. A vector capable of being introduced into a stem cell comprising anucleic acid sequence which produces Akt kinase activity upon expressionin a hematopoietic cell.
 2. The vector of claim 1, wherein the Aktkinase activity is activated by localization to the plasma membrane ofthe cell in which it is produced.
 3. A retroviral vector capable oftransducing hematopoietic stem cells comprising a nucleic acid sequencewhich encodes Akt kinase activity.
 4. The vector of claim 3, wherein theakt nucleic acid sequence is derived from human or mouse sequences. 5.The vector of claim 3, wherein the akt nucleic acid sequence encodes anAkt variant having altered kinase activity.
 6. The vector of claim 5,wherein the encoded Akt variant has reduced or eliminated kinaseactivity.
 7. The vector of any one of claims 3-5, wherein the aktnucleic acid further comprises sequences encoding amyristoylation/palmitylation motif which targets Akt to the plasmamembrane of a cell in which it is expressed.
 8. The vector of claims 1or 3, wherein expression of akt activity is inducible.
 9. The vector ofclaims 1 or 3, wherein plasma membrane localization of akt kinaseactivity is inducible.
 10. The vector of claims 1 or 3, wherein plasmamembrane localization of akt kinase activity remains subject tophysiological regulation.
 11. The vector of claims 1 or 3, whereinsequences encoding the akt kinase activity are flanked by sequenceswhich mediate excision from DNA upon expression of a cognaterecombinase.
 12. A vector capable of being introduced into ahematopoietic stem cell comprising a nucleic acid sequence whichproduces eIF4E, bcl-2, PTEN or eIF3E activity upon expression in ahematopoietic cell.
 13. A retroviral vector capable of transducinghematopoietic stem cells comprising a nucleic acid sequence whichencodes eIF4E, bcl-2, PTEN or eIF3E activity.
 14. The vector of claims13, wherein the eIF4E, bcl-2, PTEN or eIF3E nucleic acid sequence isderived from human or mouse sequences.
 15. The vector of claims 3 or 13,wherein the vector is lentiviral.
 16. The vector of claims 3 or 13,selected from the group consisting of MMLV and MSCV.
 17. The vector ofclaims 3 or 13, wherein the vector is MSCV.
 18. The vector of claims 1,3, 12 or 13, wherein the vector further comprises one or more additionalgenes capable of expression.
 19. The vector of claim 18, whereinexpression of one or more additional genes transforms lymphocytes intolymphoma cells.
 20. The vector of claim 19, wherein an additional geneencodes myc.
 21. The vector of claim 20, wherein myc is expressed from astrong viral transcription control sequence.
 22. The vector of claim 18,wherein an additional gene is a marker gene.
 23. The vector of claim 22,wherein the marker gene is fluorescent.
 24. The vector of claim 23,wherein the marker gene is GFP.
 25. The vector of claim 22, wherein themarker gene is selectable in the cell in which akt, eIF4E, bcl-2, PTENor eIF3E is expressed.
 26. The vector of claim 18, wherein an additionalgene is transcriptionally linked to the akt, eIF4E, bcl-2, PTEN or eIF3Enucleic acid sequence.
 27. The vector of claim 26, wherein theadditional gene is translated from an internal ribosome entry site(IRES).
 28. A retroviral vector comprising a nucleic acid sequence thatencodes akt, bcl-2, eIF4E, PTEN or eIF3E activity and further comprisinga transcriptionally linked nucleic acid sequence that encodes anIRES-gene 2 sequence.
 29. A vector comprising a sequence selected frompMSCV-akt-IRES-gene 2; pMSCV-eIF4E-IRES-gene 2; pMSCV-bcl-2-IRES-gene 2;pMSCV-PTEN-IRES-gene 2; and pMSCV-eIF3E-IRES-gene
 2. 30. A vectorcomprising a pMSCV-akt-IRES-gene 2 or pMSCV-eIF4E-IRES-gene 2 sequence.31. The vector of claims 29 or 30, wherein gene 2 encodes a selectablemarker.
 32. The vector of claims 29 or 30, wherein gene 2 encodes GFP.33. The vector of claims 29 or 30, wherein gene 2 encodes myc.
 34. Acell transduced with the vector of any one of claims 1-33.
 35. The cellof claim 34, wherein the cell further comprises an activated myc gene.36. The cell of claims 34 or 35, wherein the cell is a stem cell ortumorigenic cell.
 37. The cell of claims 34 or 35, wherein the cell is arodent cell.
 38. The cell of claims 34 or 35, wherein the cell is amouse cell.
 39. A mammal bearing a cell of any one of claims 34-38. 40.The mammal of claim 39, selected from a primate, rat or mouse.
 41. Amethod for testing a hematopoietic tumor for sensitivity to a treatment,comprising: (a) administering said treatment to a mouse bearing apopulation of hematopoietic cells comprising activated akt, bcl-2, eIF3Eor eIF4E activity or repressed PTEN activity, wherein the mouse has beenengineered to develop a hematopoietic tumor; and (b) monitoring themouse for onset of primary tumor formation, wherein increased time totumor onset indicates sensitivity to the treatment.
 42. The method ofclaim 41, further comprising the steps, after step (b), of c)administering a treatment to the tumor bearing mice which causesremission; and d) monitoring the length of time until relapse, whereinincrease time of tumor relapse indicates sensitivity of secondary tumorsto the treatment.
 43. The method of claim 41, wherein the mousecomprises a population of cells engineered to express myc from a strongviral promoter thereby resulting in the development of hematopoietictumor cells.
 44. The method of claim 43, the mouse comprising a myc geneoperably linked to an Eμ IgH enhancer.
 45. The method of claim 41,wherein activated myc, akt, bcl-2 or eIF4E activities are introduced,individually or in any combination, into stem cells or tumorgenic cellsof the mouse by means of a retroviral vector prior to treatment.
 46. Themethod of claim 41, wherein the mouse bears a cell of any one of claims34-38.
 47. A method for testing a hematopoietic tumor for sensitivity toa treatment, comprising (a) administering said treatment in vitro tohematopoietic tumor cells originally arising in a mouse engineered todevelop a hematopoietic tumor and bearing a population of hematopoieticcells comprising activated akt, bcl-2, eIF3E or eIF4E activity orrepressed PTEN activity; and (b) monitoring the cells for growth,wherein slowing or arresting of growth indicates sensitivity of thelymphoma to said treatment.
 48. The method of claim 47, the mousecomprising a population of cells engineered to express myc from a strongviral promoter thereby resulting in the development of hematopoietictumors.
 49. The method of claim 47, wherein activated myc, akt, bcl-2,eIF3E or eIF4E activities or repressed PTEN activity are introduced,individually or in any combination, into stem cells or tumorigenic cellsof the mouse by means of a retroviral vector prior to treatment.
 50. Themethod of claim 47, wherein the mouse bears a cell of any one of claims34-38.
 51. A method for testing a hematopoietic tumor for sensitivity toa treatment, comprising: a) allowing tumors to arise in recipient miceengineered to develop a hematopoietic tumor or tumor cells and bearing apopulation of hematopoietic cells comprising activated akt, bcl-2, eIF3Eor eIF4E activity or repressed PTEN activity; b) administering thetreatment to recipient mice; and c) monitoring treated recipient micefor remission of the tumor or tumor cells, wherein more frequentremissions among treated recipient mice compared to untreated recipientmice indicates sensitivity of the lymphomas to the treatment.
 52. Themethod of claim 51, the mouse comprising a population of cellsengineered to express myc from a strong viral promoter thereby resultingin the development of lymphomas.
 53. The method of claim 51, whereinactivated or repressed activities are introduced, individually or in anycombination, into stem cells or tumorigenic cells of the mouse by meansof a retroviral vector prior to the treatment.
 54. The method of claim53, wherein the stem cell is a hematopoietic stem cell.
 55. The methodof claim 51, wherein the mouse bears a cell of any one of claims 34-38.56. A method for identifying a treatment which increaseschemosensitivity of a tumor cell, comprising a) administering a testtreatment to a mouse engineered to develop a tumor or tumor cells andbearing a population of cells comprising an activated akt, bcl-2, eIF3Eor eIF4E activity or repressed PTEN activity, said mouse, aftertreatment, referred to as a test mouse; b) allowing said test mouse todevelop a tumor or tumor cells; and c) assessing the extent to which thetumor or tumor cells are present in the test mouse after the treatmentand comparing it to the extent to which such tumors or tumor cells arepresent in a control mouse; wherein if remission from the tumor or tumorcells occurs more frequently in the test mouse than in the controlmouse, the treatment is one which increases chemosensitivity.
 57. Themethod of claim 56, the mouse comprising a population of cellsengineered to express myc from a strong viral promoter thereby resultingin the development of hematopoietic tumors.
 58. The method of claim 56,wherein the activated or repressed activity is introduced, individuallyor in any combination, into stem cells or tumorigenic cells of the mouseby means of a retroviral vector prior to the treatment.
 59. The methodof claim 56, wherein the stem cells are hematopoietic.
 60. The method ofclaim 56, wherein the mouse bears a cell of any one of claims 34-38. 61.A method for detecting a genetic alteration in a tumor or tumor cell,said alteration associated with resistance to a treatment which in theabsence of alteration causes remission of the tumor or tumor cell, saidmethod comprising: a) harvesting tumor cells from a mouse engineered todevelop a hematopoietic tumor and bearing a population of cellscomprising an activated akt, bcl-2, eIF3E or eIF4E activity or repressedPTEN activity; b) transplanting the tumor cells of step a) intorecipient mice; c) allowing tumors to arise in the recipient mice; d)administering the treatment to the recipient mice, thereby achievingremission; e) monitoring the recipient mice for relapse followingremission; f) harvesting tumor cells from the recipient mice; g)optionally repeating steps b)-f) in sequence until they have beenrepeated 0, 1 or more times, wherein the recipient mice are differentmice with each repetition of steps b)-f); and h) identifying adifference in the level or function of a gene product between the tumorcells of the last performed step f) and the tumor cells of step a), saiddifference being indicative of a genetic alteration in the tumor cellsor tumors.
 62. The method of claim 61, wherein step h) of identifying adifference in the level or function of a gene product is performed usingarrays designed to monitor RNA expression.
 63. The method of claim 61,wherein step h) of identifying a difference in the level or function ofa gene product is performed using arrays designed to monitor proteinexpression.
 64. The method of claim 61, wherein the mouse in step a)comprises a population of cells engineered to express myc from a strongviral promoter thereby resulting in the development of lymphomas. 65.The method of claim 61, wherein activated akt, bcl-2, eIF3E or eIF4Eactivity or repressed PTEN activity is introduced, individually or incombination, into stem cells or tumorigenic cells of the mouse by meansof a retroviral vector prior to the treatment.
 66. The method of claim65, wherein the stem cells are hematopoietic.
 67. The method of claim61, wherein the mouse in step a) bears a cell of any one of claims34-38.
 68. The method of claim 61, further comprising the step ofidentifying the detected genetic alteration.
 69. A method for obtainingtumor cell samples of well-defined genotype, said method comprising thestep of: a) harvesting tumor cells from a mouse engineered to develophematopoietic tumors and bearing a population of cells comprising anactivated akt, bcl-2, eIF3E or eIF4E activity or repressed PTENactivity.
 70. The method of claim 69, wherein the mouse in step a)comprises a population of cells engineered to express myc from a strongviral promoter thereby resulting in the development of lymphomas. 71.The method of claim 69, wherein activated or repressed activities areintroduced, individually or in any combination, into stem cells ortumorigenic cells of the mouse by means of a retroviral vector prior toharvesting tumor cells.
 72. The method of claim 69, wherein the mouse instep a) bears a cell of any one of claims 34-38.
 73. The method of claim69, further comprising the steps of: b) transplanting the tumor cells ofthe previously performed step into recipient mice; c) allowing tumors toarise in the recipient mice; d) administering a treatment to therecipient mice, thereby achieving tumor remission; e) harvesting tumorcells from the recipient mice of e); and g) optionally repeating stepsb)-e) in sequence until they have been repeated 0, 1 or more times,wherein the recipient mice are different mice with each repetition ofsteps b)-e).
 74. A method for obtaining hematopoietic tumor cells forthe study of drug resistance associated with a gene, said methodcomprising: a) administering a treatment to mice bearing a population ofcells having a genome comprising a myc gene operably linked to an Eμ IgHenhancer, and further comprising the gene by insertion into the genomeof a pMSCV vector bearing the gene, wherein said mice develop ahematopoietic tumor or premalignant condition, the treatment therebycausing remission in the mice; b) monitoring the mice of step a) forrelapse, thereby identifying relapsed mice; c) repeating steps a) and b)in sequence until they have been repeated 0, 1 or more times; and d)harvesting lymphoma cells from the mice of the last performed step b).75. A method for producing isolated tumor cells of well-definedgenotype, said method comprising: a) isolating primary malignant cellsfrom a hematopoietic malignancy arising in a mouse bearing a populationof cells having a genome comprising a myc gene operably linked to an EμIgH enhancer, and further comprising one or more genes selected from thegroup consisting of an akt, bcl-2, eIF3E, eIF4E or PTEN gene, said geneinserted into the genome on a pMSCV vector.
 76. The method of claim 75,further comprising the steps of: b) transplanting the primary malignantcells into mice; c) allowing a hematopoietic malignancy to arise in themice of b); d) administering an anti-tumor treatment to the mice of c),thereby causing remission; e) allowing lymphomas to arise in the mice ofd); and f) isolating tumor cells from the mice of e).
 77. A method fortesting for an effect of a treatment on tumor growth, comprising: a)transducing Eμ-myc hematopoietic stem cells or tumor cells with a vectorcomprising MSCV-akt, bcl-2, eIF3E, eIF4E or PTEN sequences; b)administering transduced cells to irradiated animals, wherein therecipient animals develop tumors; c) harvesting a tumor from anirradiated animal; d) administering cells derived from the tumor to arecipient animal; e) administering a treatment to the recipient animal;and f) monitoring the recipient animal for the effect of the treatmenton tumor growth; wherein, if tumor growth is decreased or increased inthe recipient animals relative to tumor growth in control animals, thereis an inhibitory or enhancing effect, respectively, of the treatment ontumor growth.
 78. A method for testing a nucleic acid for its effect ontumorigenesis, said method comprising: a) irradiating a recipientmammal; b) introducing a test nucleic acid into stem cells ortumorigenic cells that produce tumors in a recipient mammal; c)reconstituting the recipient mammal with said altered stem cells ortumorigenic cells wherein said recipient mammal further comprises anucleic acid encoding an activated akt, bcl-2, eIF3E or eIF4E orrepressed PTEN activity, alone or in any combination; and d) observingthe effect of the test nucleic acid on tumorigenesis in the recipientmammal.
 79. The method of claim 78, wherein the stem cells ortumorigenic cells are engineered to produce hematopoietic tumors in amammal by means of an activated myc gene.
 80. The method of claim 78,wherein the test nucleic acid is introduced into a stem cell ortumorigenic cell before or in combination with a nucleic acid encodingactivated myc.
 81. The method of claim 78, wherein the test nucleic acidis introduced into a non-myc-activated stem cell or tumorigenic cellderived from a patient.
 82. The method of claim 78, wherein the testnucleic acid is co-expressed with activated akt, bcl-2, eIF3E or eIF4Eactivity or repressed PTEN activity.
 83. The method of claim 78, whereinthe test nucleic acid is introduced into the stem cells or tumorigeniccells as a member of a collection of nucleic acid molecules contained ina library.
 84. A method of inhibiting growth of an akt-, bcl-2, eIF3E-,eIF4E activated tumor cell in a mammal, said method comprisingadministering to the mammal an effective dose of rapamycin or activeanalog thereof and a chemotherapeutic agent.
 85. The method of claim 84,wherein the chemotherapeutic agent is adriamycin, cyclophosphamide ordoxorubicin.
 86. A method for assessing the sensitivity of a tumor to atest chemotherapeutic agent, said method comprising a) determiningwhether the tumor expresses activated akt, bcl-2, eIF3E or eIF4Eactivity or repressed PTEN activity; and b) treating a tumor whichexpresses said activated or repressed activity with rapamycin or anactive analog thereof in combination with the test chemotherapeuticagent; wherein the tumor is sensitive to the test chemotherapeutic agentif regression or remission occurs following said treatment.
 87. Themethod of claim 86, wherein the test chemotherapeutic agent is a DNAmodifying agent selected from the group consisting of DNA damagingagents, DNA cross-linking agents, DNA alkylating agents andtopoisomerase inhibitors that cause DNA damage.
 88. The method of claim87, wherein the agent is cyclophosphamide, melphalan, doxorubicin ordaunorubicin.
 89. The method of claim 86, wherein the testchemotherapeutic agent is expressed as a member of a nucleic acid orchemical library.
 90. A method for inhibiting the growth of akt, bcl-2,eIF3E or eIF4E activated or PTEN repressed tumors in a mammal, saidmethod comprising administering to the mammal an effective dose of aninhibitor of translation in combination with an effective dose of one ormore chemotherapeutic agents.
 91. A method for testing one or moreagent(s) for targeting a component of the PI3K/Akt pathway, said methodcomprising: immunoblot analysis for markers of akt activation selectedfrom the group consisting of: akt, p-akt, TSC1/2, p-TSC1/2, Rheb,p-Rheb, mTOR, p-mTOR, s6 kinase, p-s6 kinase, S6, p-S6, 4E-BP, p-4E-BP,eIF4G, p-eIF4G, eIF4B, p-eIF4B, bcl-2, p-bcl-2, FKHR, p-FKHR, bad,p-bad, GSK, p-GSK, eIF2B, p-eIF2B, eIF4E, p-eIF4E.
 92. A method foridentifying a potential target for drug therapy in the treatment of anakt, bcl-2, eIF3E or eIF4E activated or PTEN repressed hematopoietictumor, the method comprising: a) harvesting hematopoietic tumor cellsfrom a mouse engineered to develop a hematopoietic tumor and bearing apopulation of cells comprising an akt, bcl-2, eIF3E or eIF4E-activatedor PTEN repressed activity; b) introducing a nucleic acid library into apopulation of the harvested cells; c) transplanting a fraction of cellscomprising one or more members of the nucleic acid library intorecipient mice; d) allowing hematopoietic tumor cells to arise intransplanted recipient mice; wherein earlier recurrence and reducedsurvival of a recipient mouse compared to control mice indicates thepresence of a potential target encoded by at least one member of theintroduced nucleic acid library.
 93. A method for identifying a drug fortreatment of an akt, bcl-2, eIF3E or eIF4E activated or PTEN repressedtumor cell or tumor, the method comprising: a) harvesting tumor cellsfrom a mouse engineered to develop a tumor or tumor cells and bearing apopulation of cells comprising an akt, bcl-2, eIF3E or eIF4E activatedor PTEN repressed activity; b) treating extracts of the tumor cells withone or more test molecules; c) monitoring the effects of said treatmenton one or more markers of the akt pathway; wherein a change in thephenotype of a marker of akt signalling in the direction of decreasedakt, bcl-2, eIF3E or eIF4E activity or increased PTEN activity indicatesthe presence of a potential drug.
 94. The method of claim 93, whereinone or more test molecules are members of a chemical library.
 95. Themethod of claim 93, wherein the marker of akt signalling is selectedfrom the group consisting of: akt, p-akt, TSC1/2, p-TSC1/2, Rheb,p-Rheb, mTOR, p-mTOR, s6 kinase, p-s6 kinase, S6, p-S6, 4E-BP, p-4E-BP,eIF4G, p-eIF4G, eIF4B, p-eIF4B, bcl-2, p-bcl-2, FKHR, p-FKHR, bad,p-bad, GSK, p-GSK, eIF2B, p-eIF2B, eIF4E, p-eIF4E.